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Regular Article |
a School of Animal and Microbial Sciences, The University of Reading, Whiteknights, Reading RG6 6AJ, United Kingdom
b School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, United Kingdom
ABSTRACT
The aim was to investigate potential interactions between FSH and intraovarian growth factors in modulating secretion of inhibin A (inh A), activin A (act A), follistatin (FS), estradiol (E2), and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized FSH-responsive phenotype is maintained. Cells from 4- to 6-mm follicles were cultured in serum-free medium containing insulin (10 ng/ml) and androstenedione (10-7 M), and effects of ovine FSH (0.037–3 ng/ml) were tested alone and in combination with insulin-like growth factors (IGF) (LR3 IGF-I analogue; 2–50 ng/ml) and epidermal growth factor (EGF; 0.1–10 ng/ml). Medium was changed every 48 h and cultures ended after 144 h, when cell number was determined. Between 48–96 h and 96–144 h, FSH promoted (P < 0.0001) increases in output of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold), with maximal responses (in parentheses) elicited by 0.33 ng/ml FSH during the final period. Higher FSH doses (1 and 3 ng/ml) gave reduced responses for each of the above hormones, whereas P4 output was maximal (3-fold) at these doses. FSH promoted a slight increase in cell number (
1.7-fold; P < 0.001). LR3 IGF-I alone markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold); this was accompanied by modest increases (P < 0.01) in P4 output (2.5-fold) and cell number (2-fold). Whereas FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower doses of LR3 IGF-I, it suppressed (P < 0.001) the response to the highest dose. EGF alone promoted a 1.7-fold increase in cell number (P < 0.001) without affecting hormone release; however, it abolished (P < 0.001) FSH-induced secretion of inh A, act A, FS, and E2. Both FSH alone and LR3 IGF-I alone dose-dependently increased the act A:FS ratio (3-fold; P < 0.005) and act A:inh A ratio (3-fold to 6-fold; P < 0.001), suggesting that both factors selectively raise activin "tone" and that this could be a key requirement for FSH and IGF-induction of follicular E2 production. This hypothesis was reinforced by the finding that addition of FS, to reduce the act A:FS ratio and sequester secreted activin, markedly suppressed (P < 0.001) FSH (3-fold)-, and LR3 IGF-I (2-fold)-induced E2 output.
activin, estradiol, follicle-stimulating hormone, follistatin, granulosa cells, growth factors, inhibin, ovary, progesterone
INTRODUCTION
Throughout the course of ovarian follicle development granulosa cells proliferate and subsequently acquire differentiated functions. These processes are under the control of both systemic and locally produced regulatory molecules whose coordinated actions contribute to normal folliculogenesis, culminating in dominant follicle selection and ovulation. The critical role of FSH in follicular development is well established and increasing evidence supports a role for various systemic growth factors, intraovarian growth factors, or both, including insulin-like growth factors I and II (IGF-I and IGF-II) and their binding proteins (IGF-BPs), epidermal growth factor (EGF) family members (including transforming growth factor alpha; TGF
), and TGFß superfamily members (including inhibins and activins) as coregulators of follicle development.
Granulosa cells are a major site of inhibin (inh), activin (act), and follistatin (FS) expression. Inhibins play a key endocrine role in the negative feedback regulation of pituitary FSH secretion, whereas activins, FS, and to a lesser extent, inhibins are firmly implicated as intraovarian autocrine/paracrine regulators of follicle function. For instance, act A has been shown to promote granulosa cell proliferation [1–3], up-regulate FSH receptor expression [4, 5], and enhance steroidogenesis and inh production [6–9]. Through its role as an activin-binding protein, FS can neutralize these actions of activins [5, 10]. It is debatable whether inhibins exert autocrine actions to modulate granulosa cell function, although there is good evidence that they have a paracrine effect on thecal cells to enhance LH-induced androgen production [11–13]. Despite the wealth of evidence supporting important intrafollicular regulatory roles for these proteins, methodological limitations have meant that limited information is available on the factors that regulate the production of intact inh/act dimers and FS by granulosa cells. The recent development of novel two-site immunoassays with improved specificity and sensitivity for these peptides has provided the research tools to address this issue.
Granulosa cells express type 1 IGF receptors [14–16] and it is well established in a range of species that IGF can act both alone, and in synergy with FSH, to modulate granulosa cell proliferation, differentiation, and steroidogenesis (bovine, [17–21]; ovine, [22, 23]; rat, [24]). It is likely that most of the IGF-I present in bovine/ovine follicles is derived from the peripheral circulation, whereas most of the IGF-II is of intrafollicular (mainly thecal) origin (sheep, [14]; cattle, [16, 25]).
EGF and its closely related homologue, TGF, are expressed by various cell types; they both interact with the same cell surface receptor (EGF receptor), which is expressed by numerous cell types including granulosa and theca cells. Exposure of granulosa cells to EGF/TGF promotes cell proliferation [26] associated with a loss of differentiated function, exemplified by a marked reduction in E2 production in vitro [27–30] and in vivo [31]. Theca cells have been identified as a key site of TGF expression in the bovine ovary and evidence suggests that TGF of thecal origin exerts a local paracrine action on neighboring granulosa cells to modulate their proliferation and responsiveness to gonadotropins and other regulatory factors [26, 32].
The primary objective of the present study was to identify potential interactions between FSH and intraovarian growth factors (IGF and EGF/TGF) in their ability to modulate the secretion of inh A, act A, FS, E2, and progesterone (P4) by bovine granulosa cells cultured under conditions in which a nonluteinized "follicular" phenotype is maintained. Initial findings revealed that treatment-induced changes in E2 secretion were accompanied by broadly parallel changes in the secretion of inh A, act A, and FS. However, closer inspection of the data revealed potentially important changes in act A:FS ratio and act A:inh A ratio in cell-conditioned media. This prompted a further study involving bio-neutralization of endogenous activin to test the hypothesis that act A mediates the stimulatory effects of FSH and IGF on E2 and inh A secretion.
MATERIALS AND METHODS
Ovaries and Granulosa Cell Harvesting
Ovaries were obtained from cattle slaughtered at random stages of the estrous cycle at an abattoir. They were transported to the laboratory at ambient temperature in sterile medium-199 (M-199) containing 1% (v/v) antibiotic-antimycotic solution (Sigma UK Ltd; Poole, Dorset, UK). After rinsing briefly in ethanol (70% v/v), selected ovaries were transferred to fresh M-199 and medium-sized follicles (4- to 6-mm diameter) lacking obvious signs of atresia were removed by dissection and placed in a Petri dish containing Dulbecco phosphate buffered saline (DPBS-). After aspirating follicular fluid using a syringe fitted with a 19-gauge needle, a slit was made in the follicle wall and the granulosa cell layer was gently disrupted with the aid of a plastic inoculation loop. Cells harvested in this manner from 100 follicles were pooled, pelleted by centrifugation (800 x g for 10 min), and resuspended in 5 ml PBS. Double-distilled water (10 ml) was added and the cells were agitated for 10 sec to lyse any red blood cells present; isotonicity was quickly restored by the addition of 10 ml of 3x concentrated PBS. The cells were then pelleted by centrifugation and resuspended in a small volume of culture medium (consisting of McCoy 5A modified medium supplemented with 1% (v/v) antibiotic-antimycotic solution, 10 ng/ml bovine insulin, 2 mM L-glutamine, 10 mM Hepes, 5 µg/ml apotransferrin, 5 ng/ml sodium selenite, 0.1% BSA, and 10-7 M androstenedione (all purchased from Sigma). Cells were counted using a hemocytometer and viability estimated by trypan blue dye-exclusion was 20%–30%.
Cell Culture
Cells were seeded at a concentration of 75 x 103 viable cells/50 µl culture medium into wells of 96-well tissue culture plates (Nunclon; Life Technologies Ltd, Paisley, Ochdonough, UK) containing 200 µl pre-equilibrated culture medium with and without treatments. Plates were incubated in a water-saturated atmosphere of 5% CO2 and 95% air at 38.5°C for an initial 48-h period. Conditioned medium (175 µl) was gently removed taking care not to aspirate the loosely adherent cell clumps. Fresh culture medium (175 µl) containing appropriate treatments was replaced before the plates were returned to the incubator. Conditioned medium was removed and fresh medium and treatments were added in this manner every 48 h for 6 days of culture; conditioned-media samples were stored at -20°C.
Preparation and Addition of Test Substances
Ovine FSH (National Institute for Diabetes and Digestive and Kidney Diseases; [NIDDK] oFSH-19SIAPP), recombinant IGF-I analogue (Long R3 IGF-I, Sigma), human recombinant EGF (Amersham Life Sciences, Little Chalfont, Bucks, UK), and human recombinant follistatin-288 (NIDDK) were dissolved in Hanks balanced salt solution (HBSS) containing 0.3% (w/v) BSA, and the stock solution was sterilized by passing it through a 0.2-µm membrane filter (Minisart; Sartorius AG, Göttingen, Germany) before further dilution in sterile culture medium. The more potent LR3 IGF-I analogue was used rather than IGF-I or IGF-II because its action is not compromised by association with endogenous IGF-BPs produced by the cells [20], thus simplifying the interpretation of experiments and permitting the use of lower treatment doses. Androstenedione (Sigma) was dissolved initially in ethanol before further dilution in culture medium. Test substances were added to duplicate wells in a volume of 25 µl, and an equal volume of culture medium alone was added to control wells.
Termination of Culture and Estimation of Viable Cell Number
At the end of the culture period conditioned media were removed and stored (-20°C). Viable granulosa cell number was determined by uptake of neutral red dye [22]. Briefly, cells were incubated for 3 h with 200 µl culture medium containing 50 µg/ml neutral red dye. The medium was removed and cells fixed with 4% paraformaldehyde for 3 min. Paraformaldehyde was removed and color developed by addition of acetic acid ethanol (1% acetic acid, 50% ethanol, 49% water). Absorbance at 540 nm (with a 600-nm reference filter) was then measured. The relationship between absorbance and cell number was determined by previously incubating known numbers of cells with neutral red and constructing a standard curve. The relationship between absorbance and cell number was linear over an appropriate range.
Immunoassays
Concentrations of inh-A were determined using the two-site immunoradiometric assay described by Knight and Muttukrishna [33]. Purified 32-kDa bovine inh [34] diluted in blank culture medium was used as a standard. The detection limit of the assay was 250 pg/ml with mean intraassay and interassay coefficients of variation (CVs) of 4.6% and 7.6%, respectively. Total act A (i.e., free and FS-bound) levels were measured using a two-site ELISA [35]. Human recombinant act A (NIBSC, Potters Bar, Herts, UK) was used as a standard and the assay sensitivity was 100 pg/ml. Intraassay and interassay CVs were 5.4% and 9.0%, respectively. Total FS levels were determined using the ELISA described by Tannetta et al. [36]. Values are expressed in terms of human recombinant-follistatin preparation provided by NIDDK. Assay sensitivity was 100 pg/ml and intraassay and interassay CVs were 6.1% and 6.2%, respectively.
Concentrations of P4 were determined by direct radioimmunoassay (RIA) as described elsewhere [37]. The detection limit was 0.1 ng/ml, and intraassay and interassay CVs were 7.7% and 10.5%, respectively. Concentrations of E2 were determined by direct RIA using a commercial kit (Biostat Diagnostic Systems, Woking, Surrey, UK), which was modified and validated for bovine granulosa cell conditioned culture medium in this laboratory. Modifications involved using the antiserum at a 10-fold lower concentration, making up E2 standards (0.78–200 pg/ml) in blank culture medium, and using an in-house sheep anti-rabbit immunoglobulin G precipitating serum to separate "bound" and "free" [125I]-E2 tracer. The detection limit of the assay was 1.5 pg/ml, and intraassay and interassay CVs were 6.4% and 9.4%, respectively.
Statistical Analysis
Two-way ANOVA was used to evaluate the effect of FSH on hormone secretion during different periods of culture and the interaction between FSH and each of the growth factors tested. Where indicated, one-way ANOVA and PLSD tests were subsequently used to make individual comparisons within a given treatment group providing the initial two-way ANOVA indicated a significant (P < 0.01) effect of that treatment. Unless stated otherwise, results presented are means ± SEM based on combined data from four to six individual cultures. With the exception of Figure 1, which shows hormone secretion data for all three culture periods, results are presented only for the final 96- to 144-h culture period, during which responsiveness to the various test substances was greatest.
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RESULTS
Effects of FSH on Hormone Secretion and Cell Number
Treatment of cells with FSH alone had a marked dose- and time-dependent stimulatory effect on hormone secretion (Fig. 1). Responsiveness was minimal during the first 48-h culture period but increased dramatically (P < 0.0001) between 48 and 96 h to maximal levels in the final culture period (96–144 h). Relative to basal hormone secretion in the final 96- to 144-h culture period, maximal responses (in parentheses) in terms of secretion of inh A (6-fold), act A (15-fold), FS (6-fold), and E2 (18-fold) were observed at an FSH dose of 0.33 ng/ml (P < 0.0001 in each case). At the higher doses tested (1 and 3 ng/ml) FSH gave reduced responses for each of the above hormones (ANOVA, P < 0.025–0.005). In contrast, maximal P4 output (only 3-fold, P < 0.001) was observed at the higher doses of FSH (1 and 3 ng/ml) tested. The calculated ED50 values (dose of FSH that gave a 50% response of maximum hormone production) were not significantly different for E2, inh A, act A, or FS (Table 1). The ED50 value for P4 was significantly (P < 0.0001) greater than for the above hormones. Treatment with FSH promoted a small though significant (1.7-fold, P < 0.0001) increase in cell number, which was determined at the end of the 144-h culture period (basal, 43 809 ± 3343 cells; 3 ng/ml; FSH, 74 961 ± 7410 cells).
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Effects of LR3 IGF-I on Basal and FSH-Induced Hormone Secretion and Cell Number
When tested in the absence of FSH, LR3 IGF-I promoted a dose- and time-dependent increase in secretion of all five hormones measured. As observed for FSH, responsiveness to LR3 IGF-I was minimal during the first period of culture and greatest during the final period of culture. As shown in Figure 2 (final culture period only), LR3 IGF-I markedly increased (P < 0.0001) output of inh A (8-fold), act A (41-fold), FS (12-fold), and E2 (18-fold), whereas relatively modest increases (P < 0.01) in P4 output (2.5-fold) and cell number (2-fold) were observed.
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Two-way ANOVA revealed a significant (P < 0.0001) interaction between the effects of FSH and LR3 IGF-I on hormone secretion. Thus, FSH enhanced inh A, act A, FS, and E2 secretion evoked by lower (submaximal) doses of LR3 IGF-I, but dose-dependently suppressed (P < 0.001) hormone secretion evoked by the highest dose of LR3 IGF-I tested (Fig. 2). There was no significant interaction between the effects of FSH and LR3 IGF-I on P4 secretion or cell number (P = 0.84 and 0.29, respectively).
Effects of EGF on Basal and FSH-Induced Hormone Secretion and Cell Number
In contrast to the effect of LR3 IGF-I, addition of EGF in the absence of FSH did not significantly affect the secretion of any of the five hormones examined; EGF alone did, however, increase cell number (P < 0.0001) 1.5-fold (Fig. 3). When tested in combination, EGF had a dose-dependent suppressive effect (P < 0.001) on FSH-induced secretion of inh A, act A, FS, and E2 but did not modify P4 secretion. Conversely, EGF and FSH had an additive stimulatory effect on cell number (maximal 2.3-fold increase with EGF + FSH compared with a 1.5-fold increase with EGF alone; P < 0.001).
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Effects of FSH and Growth Factors on Activin "Tone" as Reflected by Act A:FS Ratio and Act A:inh A Ratio
The ratio of act A concentration in cell-conditioned media to that of its binding protein, FS (act A:FS ratio) and the ratio of act A concentration to that of its functional antagonist, inh A (act A:inh A ratio) were calculated to provide an indication of relative activin "tone" and to determine whether this varied according to treatment. As shown in Figure 4, treatment of cells with FSH alone promoted dose-dependent (3-fold) increases in the act A:FS ratio (P < 0.0001) and the act A:inh A ratio (P < 0.0001). Treatment with LR3 IGF-I alone gave corresponding increases of 3-fold (P < 0.002) and 6-fold (P < 0.0001).
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Two-way ANOVA revealed a significant (P < 0.0001) interaction between the effects of FSH and LR3 IGF-I on act A:inh A ratio. Whereas FSH enhanced act A:inh A ratio in the presence of the lower doses of LR3 IGF-I tested, it suppressed (P < 0.001) the act A:inh A ratio in the presence of the highest dose of LR3 IGF-I (Fig. 4). This treatment x treatment interaction was not evident in the case of the act A:FS ratio. In contrast to FSH and LR3 IGF-I, EGF treatment did not significantly affect the act A:FS ratio (P = 0.134). However, EGF promoted a modest, though significant, increase in the act A:inh A ratio (1.7-fold, P < 0.001).
Effects of FS on Basal, FSH- and LR3 IGF-I-Induced E2 and Inhibin A Secretion
As shown in Figure 5, addition of an excess of FS (200 ng/ml) to sequester endogenous activin secreted by the cells resulted in a marked reduction in basal and FSH-induced secretion of E2 (P < 0.001) and inh A (P < 0.0001). Likewise, FS reduced LR3 IGF-I-induced secretion of both E2 (P < 0.02) and inh-A (P < 0.001).
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DISCUSSION
In contrast to numerous studies in which bovine granulosa cells were cultured under conditions that promote rapid luteinization and consequent loss of FSH-responsiveness and aromatase activity [38–43], cells cultured using the present system, first developed by Gutierrez et al. [20], were highly responsive to FSH in terms of induction of E2 secretion. Moreover, as we show for the first time, the cells also responded to FSH with concomitant increases in the secretion of inh A, act A, and FS, proteins that have also been implicated in the regulation of follicle growth through both systemic actions, intraovarian actions, or both [10, 44]. We are not aware of any previous granulosa cell studies on any species in which parallel measurements of the secretion of steroids (E2 and P4) and each of the three inhibin-related proteins (inh A, act A, and FS) have been recorded in the same series of experiments. Indeed, immunoassays of the required specificity and sensitivity to permit this approach have only become available in the last few years.
The observed effects of FSH, IGF, and EGF on granulosa cell steroid production and cell proliferation are generally consistent with previous findings (bovine, [20, 45]; ovine, [22]; rat, [46]) and support the utility of this serum-free culture system as a valid model system for studying other aspects of bovine follicle function, including the production and local action(s) of inhibin-related proteins. In contrast to the present findings in the bovine, it was reported [22] that FSH-induced E2 secretion by sheep granulosa cells was not accompanied by an increase in ir-inhibin secretion. Similarly, Klein et al. [47] reported that FSH promoted a 2-fold to 3-fold increase in FS secretion by bovine granulosa cells but did not affect ir-inhibin secretion. These discrepancies most likely reflect the fact that we used an assay that specifically detects dimeric inh A (-ßA dimer), rather than subunit-directed RIAs as used by these authors. In agreement, Boudjemaa et al. [48] recently reported an FSH-induced increase in inh A secretion by bovine granulosa cells cultured in conditions similar to ours; they also used a two-site immunoassay to specifically detect the -ßA dimer.
The synergistic effect of FSH and IGF on secretion of estradiol and inhibin-related proteins we observed illustrates both the potential importance and the complexity of the interplay between gonadotropins and intraovarian growth factors. Augmentation by IGF of FSH-induced E2 secretion has been reported previously in bovine [21, 49], ovine [22], and rat [24], but their concomitant effects on inh A, act A, and FS have not been investigated before now. It is interesting that augmentation of FSH action by IGF was observed only at low-dose combinations of FSH and IGF. The observed decline in FSH-stimulated production of E2, inh A, act A, and FS when high doses of FSH were added alone or in the presence of high doses of IGF appears to mimic the physiological situation in vivo because granulosa cell luteinization, initiated by exposure to the preovulatory gonadotropin surge is associated with a marked decline in expression of aromatase, inhibin/activin subunits, and FS in cattle [50, 51]. Moreover, P4 output was greatest when cells were stimulated with high doses of FSH and LR3 IGF-I (above those needed for optimum secretion of E2 and inhibin-related proteins), which is consistent with the onset of luteinization.
In contrast to LR3 IGF-I, treatment of cells with EGF alone had no effect on hormone secretion but its well-established mitogenic action [52, 53] was clearly evident from a dose-dependent increase in cell number. Moreover, EGF profoundly suppressed FSH-induced secretion of E2, inh A, act A, and FS but did not affect FSH-induced P4 secretion. Inhibitory effects of EGF/TGF on E2 production in vitro have been observed in sheep [22], human [28], and rat [29] granulosa cell cultures and in intact mouse follicle cultures [30]. In addition, EGF and TGF have also been shown to acutely suppress follicular steroid output when infused into the sheep ovary in vivo [54, 55]. To our knowledge, effects of EGF/TGF on granulosa cell production of dimeric inhibin, activin, and FS have not been examined previously. Although TGF of thecal origin is probably the main intrafollicular ligand for granulosa cell EGF receptors in the bovine [32, 45], our observation that its close homologue, EGF, uniformly suppressed FSH-induced secretion of four distinct granulosa cell markers (E2, inh A, act A, and FS) further supports the view that paracrine growth factors secreted by theca cells exert paracrine effects on granulosa cells to modulate both their proliferation and gonadotropin-dependent function.
The intrafollicular ratio of act A:FS and of act A:inh A are potentially important parameters to consider; FS functions as a high-affinity binding protein for act A and can abolish its bioactivity [56, 57] and inh A opposes the actions of act A. Hence, the ratio of act A to either FS or inh A is likely to reflect how much unopposed activin is available to interact with activin receptors on follicular cells (i.e., its "activin tone"). It is notable that treatment with FSH alone, IGF alone, and with FSH and IGF in low-dose combinations, increased the ratio of act A:FS and of act A:inh A in cell-conditioned media, suggesting that these treatments raised "activin tone" relative to that in unstimulated control cells.
Activin is a known stimulator of E2 production in granulosa cells via enhancement of FSH receptor expression and of FSH-induced aromatase activity [4, 5, 58]. Activin also up-regulates granulosa cell expression of inhibin subunit mRNA and FS mRNA [10]. Therefore, the question was posed: Does FSH-induced activin actually mediate the stimulatory effects of FSH and IGF on secretion of E2 and inhibin-related proteins? To address this issue we added an excess of exogenous FS to sequester endogenous activin produced by the cells and artificially reduce the act A:FS ratio. This resulted in significant suppression of both FSH- and IGF-stimulated secretion of both E2 and inh A by cells, suggesting that the ability of both tropic factors to promote follicular E2 and inh A production is indeed dependent, at least in part, on an induced increase in activin tone. However, FS is known to bind to certain other TGFß superfamily members apart from activin, including several of the bone morphogenic proteins (BMPs) [59]; we cannot exclude the possibility that the observed effect of exogenous FS is due to the neutralization of some other TGFß-related peptide produced by the cells.
It is possible that the FSH- and/or IGF-induced increase in granulosa cell activin tone reflects an autocrine positive feedback mechanism to up-regulate FSH and/or IGF receptor expression and thus enhances responsiveness to FSH and/or IGF [60]. Alternatively, raised activin tone may enhance E2 and inh A secretion through one or more additional intracellular mechanisms that are independent of FSH/IGF receptor up-regulation. Regardless of the mechanism of action, these findings are consistent with previous in vitro evidence implicating activin as a factor that delays the onset of atresia and premature luteinization of bovine granulosa cells from preovulatory follicles, as indicated by a reduction in secretion of P4 and oxytocin [61]. Likewise, it supports evidence that FS favors bovine follicle atresia, luteinization, or both through antagonism of activin action [9]. However, it should be noted that in contrast to the present studies, Shukovski and colleagues [9, 61] used fully differentiated (LH-responsive) granulosa cells from preovulatory bovine follicles that were cultured in medium supplemented with a supraphysiological concentration of insulin (0.3 µg/ml). Other studies indicate that such cells are essentially unresponsive to FSH and IGF in terms of induction of E2 and inhibin secretion [37, 20] and, in contrast to the present study, Shukovski and colleagues [9, 61] did not attempt to evaluate FSH-induced hormone secretion.
Within rat antral follicles, IGF-I mRNA is preferentially expressed in granulosa cells that line the antrum and surround the oocyte [62, 63]. It was suggested that this might imply a paracrine role for IGF-I in regulating oocyte maturation and indeed, Herrier et al [64] observed a stimulatory effect of IGF-I on in vitro developmental competence of cumulus-enclosed bovine oocytes. This action is presumably mediated by type 1 IGF-I receptors on granulosa cells. In view of recent findings that activin can increase oocyte maturation and developmental competence [65–67] in an FS-reversible manner [67] it is speculated that IGF-I indirectly affects oocyte maturation by raising activin tone of the surrounding cumulus cells. A comparable positive action of theca-derived IGF-II (the principle source of IGF in the bovine follicle; [16]) on cumulus cell activin tone seems equally plausible.
In summary, this study has used an improved serum-free culture system together with a panel of sensitive and specific immunoassays to provide further information on the intrafollicular actions of the growth factors IGF and EGF/TGF in modulating FSH-induced granulosa cell function (i.e., steroidogenesis, production of inhibin-related proteins, and cell proliferation) in cattle. Although IGF can potentiate the stimulatory action of FSH on cellular differentiation as shown by enhanced steroid and peptide hormone secretion, EGF/TGF promotes cell proliferation, accompanied by a profound reduction in FSH-induced secretion of E2 and inhibin-related proteins. This study also provides evidence that FSH and IGF increase the activin tone of granulosa cells and that at least some of the stimulatory actions of FSH and IGF on E2 and inh A secretion are mediated by locally produced activin acting in an autocrine/paracrine manner. These observations taken together highlight the complex nature of gonadotropin and intraovarian growth factor comodulation of folliculogenesis and suggest a previously unrecognized activin-dependent component to the action of FSH and IGF on granulosa cell function that warrants further investigation.
ACKNOWLEDGMENTS
We thank Mr. S.A. Feist for collecting bovine ovaries, Dr. G.S. Pope for providing progesterone antiserum, and Dr. P. Smith (NIDDK) for providing ovine FSH and recombinant human follistatin.
FOOTNOTES
First decision: 10 April 2001.
1 This work was supported by the Biotechnology and Biological Sciences Research Council of Great Britain (grant 45/SO5760 to P.G.K.). 
2 Correspondence. FAX: 44 118 931 0180; p.g.knight{at}reading.ac.uk
Accepted: May 8, 2001.
Received: March 14, 2001.
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M. Mihm, P.J. Baker, J.L.H. Ireland, G.W. Smith, P.M. Coussens, A.C.O. Evans, and J.J. Ireland Molecular Evidence That Growth of Dominant Follicles Involves a Reduction in Follicle-Stimulating Hormone Dependence and an Increase in Luteinizing Hormone Dependence in Cattle Biol Reprod, June 1, 2006; 74(6): 1051 - 1059. [Abstract] [Full Text] [PDF]
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 C. Glister, N. P Groome, and P. G Knight Bovine follicle development is associated with divergent changes in activin-A, inhibin-A and follistatin and the relative abundance of different follistatin isoforms in follicular fluid J. Endocrinol., February 1, 2006; 188(2): 215 - 225. [Abstract] [Full Text] [PDF]
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J Buratini Jr, A B Teixeira, I B Costa, V F Glapinski, M G L Pinto, I C Giometti, C M Barros, M Cao, E S Nicola, and C A Price Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles Reproduction, September 1, 2005; 130(3): 343 - 350. [Abstract] [Full Text] [PDF]
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O J Ginther, E L Gastal, M O Gastal, and M A Beg In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares Reproduction, April 1, 2005; 129(4): 489 - 496. [Abstract] [Full Text] [PDF]
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C. Glister, S. L. Richards, and P. G. Knight Bone Morphogenetic Proteins (BMP) -4, -6, and -7 Potently Suppress Basal and Luteinizing Hormone-Induced Androgen Production by Bovine Theca Interna Cells in Primary Culture: Could Ovarian Hyperandrogenic Dysfunction Be Caused by a Defect in Thecal BMP Signaling? Endocrinology, April 1, 2005; 146(4): 1883 - 1892. [Abstract] [Full Text] [PDF]
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I. Demeestere, C. Gervy, J. Centner, F. Devreker, Y. Englert, and A. Delbaere Effect of Insulin-Like Growth Factor-I During Preantral Follicular Culture on Steroidogenesis, In Vitro Oocyte Maturation, and Embryo Development in Mice Biol Reprod, June 1, 2004; 70(6): 1664 - 1669. [Abstract] [Full Text] [PDF]
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O.J. Ginther, E.L. Gastal, M.O. Gastal, and M.A. Beg Critical Role of Insulin-Like Growth Factor System in Follicle Selection and Dominance in Mares Biol Reprod, May 1, 2004; 70(5): 1374 - 1379. [Abstract] [Full Text] [PDF]
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C. Glister, C F. Kemp, and P. G Knight Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin Reproduction, February 1, 2004; 127(2): 239 - 254. [Abstract] [Full Text] [PDF]
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T. Fayad, V. Levesque, J. Sirois, D. W. Silversides, and J. G. Lussier Gene Expression Profiling of Differentially Expressed Genes in Granulosa Cells of Bovine Dominant Follicles Using Suppression Subtractive Hybridization Biol Reprod, February 1, 2004; 70(2): 523 - 533. [Abstract] [Full Text] [PDF]
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O.J. Ginther, D.R. Bergfelt, M.A. Beg, C. Meira, and K. Kot In Vivo Effects of an Intrafollicular Injection of Insulin-Like Growth Factor 1 on the Mechanism of Follicle Deviation in Heifers and Mares Biol Reprod, January 1, 2004; 70(1): 99 - 105. [Abstract] [Full Text] [PDF]
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 S. M. Quirk, R. G. Cowan, R. M. Harman, C.-L. Hu, and D. A. Porter Ovarian follicular growth and atresia: The relationship between cell proliferation and survival J Anim Sci, January 1, 2004; 82(13_suppl): E40 - 52. [Abstract] [Full Text] [PDF]
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F. Jimenez-Krassel, M. E. Winn, D. Burns, J. L. H. Ireland, and J. J. Ireland Evidence for a Negative Intrafollicular Role for Inhibin in Regulation of Estradiol Production by Granulosa Cells Endocrinology, May 1, 2003; 144(5): 1876 - 1886. [Abstract] [Full Text] [PDF]
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C. Glister, N. P. Groome, and P. G. Knight Oocyte-Mediated Suppression of Follicle-Stimulating Hormone- and Insulin-Like Growth Factor-Induced Secretion of Steroids and Inhibin-Related Proteins by Bovine Granulosa Cells In Vitro: Possible Role of Transforming Growth Factor {alpha} Biol Reprod, March 1, 2003; 68(3): 758 - 765. [Abstract] [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, K. Kot, C. Meira, and D.R. Bergfelt Associated and Independent Comparisons Between the Two Largest Follicles Preceding Follicle Deviation in Cattle Biol Reprod, February 1, 2003; 68(2): 524 - 529. [Abstract] [Full Text] [PDF]
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O.J. Ginther, C. Meira, M.A. Beg, and D.R. Bergfelt Follicle and Endocrine Dynamics During Experimental Follicle Deviation in Mares Biol Reprod, September 1, 2002; 67(3): 862 - 867. [Abstract] [Full Text] [PDF]
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O.J. Ginther, M.A. Beg, D.R. Bergfelt, and K. Kot Activin A, Estradiol, and Free Insulin-Like Growth Factor I in Follicular Fluid Preceding the Experimental Assumption of Follicle Dominance in Cattle Biol Reprod, July 1, 2002; 67(1): 14 - 19. [Abstract] [Full Text] [PDF]
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