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a CSIRO Division of Animal Production, Delivery Centre, Blacktown, New South Wales 2148, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The FGF family consists of at least 9 peptide growth factors that function through 4 known high-affinity cell-surface receptors (Kd 10–100 pM) with intrinsic tyrosine kinase activity, in common with many growth factor receptors [6, 7]. In addition to binding to high-affinity receptors, FGFs also bind to low-affinity (Kd 2–10 nM) receptors [8].
Other autocrine/paracrine agents such as the IGFs are associated with molecules with the capacity to regulate their actions at the target cell surface. The FGFs have been associated with heparin since it was found to enhance the effects of FGFs (contained in crude tumor angiogenesis factor) on capillary growth, but was inactive on its own [9]. Heparin has a strong binding affinity for FGFs [10] and is regularly used for their purification. Glycosaminoglycans (GAGs) with structures similar to that of heparin are present in extracellular matrix and on cell surfaces. These molecules were originally thought to simply protect FGFs from degradation, but have more recently been shown to be an obligatory part of the interaction of FGFs with high-affinity FGF receptors [11, 12]. Thus GAGs may act as low-affinity cell-surface receptors for FGFs, enhancing FGF binding to adjacent high-affinity tyrosine kinase receptors [12]. GAGs have also been shown to be present in the avian ovary [13]; consequently, there is clear potential for these molecules to regulate the ovarian actions of FGFs in chicken preovulatory follicles.
In the mammalian ovary, FGF is known to be secreted by preovulatory follicles and to have mitogenic effects on granulosa cells [14, 15]. Additionally FGF-2 mRNA is present in rat ovaries [16]. The occurrence of FGFs in the avian ovary is unknown, but the evidence from mammalian studies suggests that there could be a role for these growth factors in avian ovarian growth or function.
The aims of this study were to establish whether the FGFs may be partly responsible for preovulatory growth of ovarian follicles and to elucidate their mechanisms of action. This was done by measuring the effects of FGFs on DNA synthesis in chicken granulosa and theca cells in vitro. Four members of the FGF family were studied (FGF-1, FGF-2, FGF-5, and FGF-7), chosen according to their availability. Secondly, experiments were conducted with FGF binding and signaling inhibitors to establish which elements of the FGF-2 cell-surface receptor system function on these cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Individually caged White Leghorn hens were fed a commercial diet ad libitum (Millmaster Feeds Pty. Ltd., Merrylands, NSW, Australia). These birds were reared under commercial restricted lighting conditions for broiler breeder hens, the light period being increased to 15.25 h daily by the beginning of lay. Hens were killed by cervical dislocation between 30 and 50 wk of age. Birds used in each study were of the same age and laying an egg each day.
Reagents
Collagenase type II, purified from Clostridium histolyticum, was obtained from Sigma Chemical Co. (Castle Hill, NSW, Australia). Medium 199 (M199) containing Earle's salts and 4.2 mM sodium bicarbonate (GIBCO, Glen Waverley, Victoria, Australia) was supplemented (s-M199) with HEPES buffer (20 mM), L-glutamine (2 mM), sodium pyruvate (2 mM), streptomycin (0.1 mg/ml), and penicillin (1000 U/ml). Fetal calf serum (FCS) was obtained from ICN Biomedicals Pty. Ltd. (Castle Hill, NSW, Australia) and was heat-inactivated by heating at 56°C for 30 min. Ovine recombinant FGF-5 was a kind gift of Dr. Robert Seymour, CSIRO Division of Animal Production, Prospect, NSW. Heparin, purified from porcine intestinal mucosa, recombinant FGF-1, FGF-2, FGF-7, and all other reagents (unless stated) were obtained from Sigma Chemical Co.
Tissue Collection and Cell Culture
In each experiment, the granulosa and theca tissues were obtained from the same hen. The granulosa and theca layers were separated from small white preovulatory follicles (3–6-mm diameter) in sterile Dulbecco's PBS, then transferred to s-M199 and dispersed with collagenase (1-mg/ml and 5-mg/ml solutions for granulosa and theca layers, respectively). Granulosa layers were incubated in collagenase for 1–5 min at room temperature before centrifugation at 400 x g for 10 min. The pelleted cells were then washed by resuspension in fresh s-M199 and collected by recentrifugation. The cells were washed twice more in fresh s-M199 medium. Theca layers were shaken gently for 45 min in a water bath at 37°C. The volume of the theca cell suspension was then made up to 5 ml with s-M199 and Percoll (Pharmacia and Upjohn Inc., Kalamazoo, MI) to produce a 40% Percoll solution (v:v). This was centrifuged at 400 x g for 10 min to separate the theca cells from red blood cells. The top layer containing the theca cells was carefully removed to a new sterile centrifuge tube, diluted with s-M199 to reduce the Percoll concentration to less than 20%, and then centrifuged to pellet the theca cells. These were washed twice in fresh s-M199.
The density and viability of cell preparations was determined using a hemocytometer and the trypan blue exclusion technique as described previously [2]. The viability of all granulosa and theca cell preparations was greater than 95% and 90%, respectively. The absence of erythrocytes from theca cell preparations was confirmed during the viability measurements. Cells were resuspended in s-M199 with 3% FCS before being plated on plastic 48-well tissue culture plates (COSTAR, Trace Biosciences, Castle Hill, NSW, Australia), each well floor having a surface area of 1 cm2. The cell suspension volume was 1 ml per well. Cells were cultured in a humidified incubator with an atmosphere of 5% CO2 at a temperature of 41°C. Granulosa and theca cells were plated at densities of 5 x 104/cm2 and 2.5 x 105/cm2 viable cells, respectively. After an undisturbed attachment period of 48 h, the medium was removed and replaced with s-M199 containing no FCS. This was repeated after a further 24 h of culture, and at this point all treatments were made (except in the heparin/FGF-2 time-course experiment, in which the heparin treatments were made 2, 4, or 6 h after the FGF-2 treatments). For the experiment in which FGF treatment was combined with heparin, heparan sulphate, or chondroitin sulphate, treatments were made simultaneously after the second medium replacement. Treatments continued for a period of 24 h, after which time the cultures were terminated.
Cell Proliferation
Granulosa and theca cells were plated on 48-well plates and allowed to attach as described above. After 48 h of culture, the medium was replaced with fresh s-M199 containing no FCS. This was repeated after a further 24 h of culture, whereupon cells were treated with FGF-1 (0.5, 1, 5, 10, or 15 ng/ml) or FGF-2 (0.1, 0.5, 1, 5, or 10 ng/ml). Control cultures were untreated. Treatments remained with the cells for 24 h, after which time they were aspirated and the cells were detached with addition of 0.25% trypsin/5 mM EDTA (125 µl) for 5 min at 37°C. Complete detachment was ensured by gently pipetting the cell suspensions after incubation. Tryptic activity was inhibited by the addition of s-M199 (375 µl). The suspensions from each well were transferred to a microcentrifuge tube, pelleted by centrifugation at 400 x g for 10 min, resuspended in fresh s-M199, pelleted again, and then resuspended in 20 µl of fresh s-M199. The cells were then counted using a hemocytometer as described above.
Blocking Tyrosine Kinase and Endogenous GAGFGF-2-Binding Activity
Genistein is an inhibitor of substrate binding to EGF and FGF receptor protein tyrosine kinases [17]. This compound was used to block tyrosine kinase receptor pathways in cultured chicken granulosa and theca cells. The objective of these experiments was to determine whether or not the mitogenic effects of FGF-2 on granulosa and theca cells are mediated via these high affinity cell-surface receptors. If not, then any increase in DNA synthesis by these cells due to FGF-2 treatment would be unaffected by treatment with genistein.
Hexadimethrine bromide was used to elucidate the role of endogenous heparin-like GAGs that may be present on the surfaces or in the extracellular matrices of cultured follicular cells. This compound is a polycation that has recently been shown to bind to cell-surface GAGs in murine cell cultures, thus preventing FGF-2 from binding to these molecules [18]. If endogenous GAGs are influential in FGF-2 ligand-receptor binding, then addition of hexadimethrine bromide to FGF-2-treated cultures should attenuate any increased DNA synthesis due to FGF-2.
The mitogenic actions of EGF are not affected by hexadimethrine bromide [18], suggesting that it may be a suitable negative control for these experiments. However, a comparison of the growth effects of EGF and transforming growth factor 
(TGF) in chicken ovarian cells [19] and a study comparing the binding of TGF and EGF to the chicken EGF receptor [20] suggests that TGF has a greater affinity for the EGF receptor than does EGF itself in chicken cells. Therefore TGF was used in preference to EGF as a negative control in this study.
Measurement of DNA Synthesis
Incorporation of [3H]thymidine by cultured cells was used as an index of DNA synthesis. Eight hours after the experimental treatments were applied, 0.25 µCi [3H]thymidine label (Amersham Australia, Castle Hill, NSW, Australia) was added to the medium in each well, and the culture was continued for a further 16 h. At the end of this period, the cultures were terminated by aspiration of the supernatant from each well. The cell monolayer was then washed three times with PBS (1 ml), and DNA was precipitated by the addition of 500 µl trichloroacetic acid (10%) for 20 min at 4°C. The insoluble fraction was dissolved in 500 µl of sodium hydroxide (0.5 M). and the plates were incubated at room temperature for 2 h with gentle agitation. The cell solution was then diluted 1:6 with scintillant (Optiphase Hisafe III; The Australian Chromatography Company, Thornleigh, NSW, Australia), and the radioactivity in the samples was measured in counts per minute (cpm) using an LKB Wallac (Turku, Finland) 1215 Rackbeta II liquid scintillation counter.
To determine the proportion of [3H]thymidine incorporation that was due to DNA synthesis and DNA repair, granulosa and theca cells were treated with FGF-1 (10 ng/ml) or FGF-2 (0.5 ng/ml) in the presence or absence of hydroxyurea (0.76 mg/ml), an inhibitor of DNA synthesis but not of DNA repair.
Statistics
Treatments were applied to quadruplicate wells in each of three replicate experiments. The results shown in the text figures are the means and standard deviation for quadruplicate wells from one experiment. Effects reported as statistically significant in representative experiments were also significant at the same confidence interval in each replicate experiment. The pattern of the responses was the same in each repeated experiment; however, the magnitudes of the responses, measured as a proportion of the baseline, were not identical for each experiment. This is a feature of primary cell culture, in which each experiment is performed using ovarian cells taken from an individual hen. The significance of differences between treatments was measured using Student's t-test (unpaired, 2-tailed) with the StatView statistical package for the Apple Macintosh computer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The [3H]thymidine incorporation (cpm/well) by granulosa and theca cell cultures that were untreated, or treated with FGF-1, FGF-2, FGF-5, or FGF-7 over a range of doses are shown in Figure 1. Treatment with FGF-1 gave a dose-dependent increase in incorporation by both granulosa and theca cell cultures with a minimal effective dose of 1 ng/ml. At 25 ng/ml the incorporation appeared to have reached a plateau in granulosa cells, but not in theca cells. Doses above 25 ng/ml were not used in this experiment; thus the maximal effective dose of FGF-1 was not determined. Treatment with FGF-2 also increased incorporation; however, the dose-response curves were different in shape, a maximal effect was reached at a dose of 0.1 ng/ml in granulosa cells and at 0.5 ng/ml in theca cells. Treatment with FGF-2 resulted in a biphasic effect: at higher doses of FGF-2, DNA synthesis declined until basal values were reached at doses above 25 ng/ml. None of the doses of FGF-5 or FGF-7 had any effect on the DNA synthesis of either granulosa or theca cell cultures.
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Concurrent treatment of granulosa or theca cells with either FGF-1 or FGF-2 and hydroxyurea resulted in a complete inhibition of the [3H]thymidine incorporation that was caused by the growth factors alone (Fig. 2a), indicating that increases in incorporation caused by these two growth factors were due to DNA synthesis and not DNA repair. Incorporation by hydroxyurea-treated cells was not significantly different from that of the untreated controls.
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Cell Proliferation
To determine whether FGF-1 and FGF-2 induce an increase in cell number as well as DNA synthesis, granulosa and theca cell cultures were treated with FGF-1 (0.5–15 ng/ml) or FGF-2 (0.1–10 ng/ml), or were untreated. Treatment with either growth factor resulted in an increase in cell numbers in both cell types (Fig. 3); the resulting dose-response curves are similar to those seen in the [3H]thymidine incorporation studies described above.
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Effect of Genistein on FGF-2-Stimulated Mitogenesis
Granulosa and theca cell cultures were either untreated or treated with genistein (4 mg/ml) with or without FGF-2 (0.5 ng/ml). When genistein was added to cells treated with FGF-2, incorporation due to FGF-2 was completely inhibited (see Fig. 2b). Treatment with genistein alone had no significant effects on either granulosa or theca cell [3H]thymidine incorporation (not shown).
Effect of GAGs on FGF-2-Stimulated Mitogenesis
To study the role of GAGs in the mitogenic action of FGF-2, chicken granulosa and theca cell cultures were treated with FGF-2 (0.5 ng/ml) with or without increasing doses of either heparin, heparan sulphate, or chondroitin sulphate. Treatment with heparin brought about a dose-dependent inhibition of FGF-induced mitogenesis in both cell types (Fig. 4a). The [3H]thymidine incorporation at the highest dose of heparin (20 µg/ml) was less than 50% of the incorporation by the control cultures, which were treated with FGF-2 alone. However, treatment with the other two GAGs had no significant effect on the actions of FGF-2 (results not shown). Treatment of cells with either of the three GAGs in the absence of FGF-2 had no effect on basal [3H]thymidine incorporation at any of the doses used in these experiments (results not shown).
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In an effort to determine whether heparin was acting at the point of FGF-receptor binding, or before this, cells were treated with FGF-2 (0.5 ng/ml) and heparin (30 µg/ml) simultaneously, or heparin was added separately at times ranging from 2 to 6 h after the FGF-2 treatment. The inhibitory effects of heparin on FGF-2-induced mitogenesis were reduced in both granulosa and theca cell cultures as the time difference in the two treatments increased (Fig. 4b). At a time difference of 6 h there was no significant difference in the [3H]thymidine incorporation between the FGF-treated controls and the heparin/FGF-2 treated cells.
Blocking of Endogenous GAGs with Hexadimethrine Bromide
Since free heparin did not enhance the effects of FGFs, hexadimethrine bromide was used to elucidate the role of endogenous heparin-like molecules that may be present on the surfaces or in the extracellular matrices of cultured follicular cells.
Cultures were set up as in the previous experiments and treated with FGF-2 (0.5 ng/ml) with or without hexadimethrine bromide (0.1–2.5 µg/ml). In both of the FGF-2/hexadimethrine bromide-treated group of cultures, [3H]thymidine incorporation was progressively inhibited by increasing doses of hexadimethrine bromide (Fig. 5). At the maximum dose of hexadimethrine bromide, FGF-2-induced [3H]thymidine incorporation by granulosa cells was reduced to 16.4 (± 1.19)% of the incorporation by the FGF-2-treated control and incorporation by theca cells to 17.1 (± 1.04)%. Hexadimethrine bromide treatment alone had no effect on the basal level of [3H]thymidine incorporation, indicating that the effect was a specific inhibition of FGF-2-induced mitogenesis (results not shown). There was no inhibition of TGF-induced incorporation by hexadimethrine bromide in the negative control cultures.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a result of recent studies with mammalian cells [11, 24], FGFs are now known to initiate their growth and differentiating effects via a complex cell-surface receptor interaction involving both high-affinity (tyrosine kinase)-type and low-affinity (GAG)-type receptors. In those studies it was found that cells that do not normally express significant levels of GAGs or that have been depleted of GAGs have been shown to be unresponsive to FGF-2 until treated with heparin. Furthermore, FGF-2 binding to high-affinity receptors on Swiss 3T3 cells is blocked by treatments that inactivate cell-surface GAGs [12].
The presence of high-affinity tyrosine kinase receptors, the first element in this FGF receptor-signaling chain, was demonstrated here by our ability to completely abolish FGF-2 actions on granulosa/theca DNA synthesis with a tyrosine kinase inhibitor, genistein.
Currently, four high-affinity FGF receptors have been characterized, FGFR-1, FGFR-2, FGFR-3, and FGFR-4 (for review see [6]). The data presented here are insufficient to show which of these receptors may be present on chicken granulosa and theca cells; however, the greater potency of FGF-2 compared with FGF-1 could rule out FGFR-3 and -4 since they are reported to have higher affinities for FGF-1 than for FGF-2 while FGFR-1 and FGFR-2 have equal affinity for these growth factors [7, 24, 25]. The latter study showed that there was no appreciable binding of FGF-5 to FGFR-2, which may further suggest that this is at least one of the FGF receptors present on chicken granulosa and theca cells. Further study will be required to elucidate the nature of the high-affinity receptors operating in chicken granulosa and theca cells.
The presence of the low-affinity type of FGF receptor in chicken cells has been suggested by work showing that heparin enhances the mitogenic effects of both FGF-1 and FGF-2 on cultured chicken adipocyte precursor cells [26], thus reducing the growth factor dose required to bring about maximal DNA synthesis. The implication of this work was that heparin was capable of reproducing the effects attributed to the low-affinity FGF receptors, GAGs, found in the extracellular matrix and on the cell membrane. Our experiments testing three GAGs (heparin, heparan sulphate, and chondroitin sulphate) for their ability to enhance FGF-2 actions showed that only heparin had any effect, and that this was inhibitory. A further experiment revealed that these inhibitory effects were progressively reduced as the time between heparin and FGF-2 treatment increased. This suggests that heparin competes for FGF-2 binding with cell-surface receptors and is consistent with a study which demonstrated that heparin inhibited FGF-2 binding to both high- and low-affinity FGF receptors [27]. While this study did not determine the presence of endogenous GAGs in ovarian cell cultures, differences in the relative abundance of these molecules in adipocyte and ovarian cell cultures may explain why exogenous heparin has opposite effects in these two systems. The hypothesis that FGF-2 is enhanced by heparin only when cells have insufficient endogenous GAGs is supported by the work of Mansukhani and colleagues [24], who showed that heparin was required for binding of FGF-2 to the FGFR-2 receptor on 32D myeloid cells (believed to be deficient in GAGs), but not on Chinese hamster ovary cells, which possess endogenous GAGs.
A further implication of our experiments is that GAGs are effective in enhancing FGF-2 actions only when they are anchored on the surface of target cells rather than being free in solution. This is consistent with a study which showed that heparin in solution could not restore the responsiveness to FGF-2 activity that was lost when cultured bovine adrenocortical cells were treated with heparitinase [28].
Since the experiments with heparin were unable to show the presence of low-affinity FGF receptors, the cell-surface GAGs of granulosa and theca cells were targeted directly with hexadimethrine bromide, which blocks the FGF binding sites on GAGs [18]. The presence of GAGs in chicken preovulatory follicles was first shown by an investigation of their role in ovulation [13]. The results of our study clearly show that hexadimethrine bromide inhibited the actions of FGF-2. There was no inhibition of the control cultures treated with TGF and hexadimethrine bromide, suggesting that this polycation had specifically targeted the FGF receptors. Thus chicken ovarian cells appear to possess cell-associated GAGs that are low-affinity FGF-2 receptors, intimating a role for GAGs in mediating the growth effects of FGFs on follicular development. Hexadimethrine bromide was unable to completely inhibit the mitogenic actions of FGF-2, even at higher doses, suggesting that a proportion of the high-affinity FGF receptors may be activated independently of the low-affinity FGF receptors in these cells. Further work to identify these receptors will be necessary to understand the nature of high- and low-affinity receptor interaction in these cells.
FGFs have been shown here to have an effect on DNA synthesis by chicken granulosa and theca cells, this growth being mediated via a complex receptor system, similar to those proposed to occur in mammalian cells [11]. These FGF-2 actions are clearly modulated by GAGs, as evidenced by the inhibitory effects of hexadimethrine bromide. A role for FGF-2 in the regulation of chicken ovarian growth is further supported by recent findings from our laboratory that demonstrate the presence of this growth factor in granulosa- and theca-conditioned culture media (unpublished data). Manipulation of this growth regulatory system by compounds such as hexadimethrine bromide may, in future, offer a means to elucidate the role that FGFs play in the rapid growth of chicken preovulatory follicles in vivo. When considered together with previous studies showning that the IGF and EGF/TGF growth factor families also have a role in the growth and differentiation of chicken ovarian follicular cells [2–4], the evidence presented here indicates that FGFs may be part of a complex ovarian growth regulation system consisting of several growth factors, associated molecules such as binding proteins and GAGs, and various types of cell-surface receptors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R.D. Roberts, Department of Animal & Poultry Science, University of Guelph, Guelph, ON, Canada N1G 2W1. FAX: 519 836 9873; rroberts{at}aps.uoguelph.ca
Accepted: July 3, 1999.
Received: December 16, 1996.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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