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Ovary |
Department of Obstetrics and Gynaecology, Reproductive Medicine Unit, The University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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androgen receptor, cumulus cells, follicular development, granulosa cells, growth factors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dihydrotestosterone (DHT), both in vivo [2, 3] and in vitro [4–7], demonstrate that androgens have a direct influence on ovarian function. This influence is potentially mediated by the androgen receptor (AR), which has been detected in ovarian cells from all vertebrate species studied to date [8–17], suggesting a conserved receptor-mediated role for androgens in folliculogenesis. Within the ovary, granulosa cells generally display the strongest AR immunoreactivity and are exposed to the most potent AR agonists, testosterone and DHT. The latter are absorbed from the secretions of thecal cells or are internally produced through enzymatic conversion of androgen precursors [18]. Therefore, granulosa cells are considered to be the main location of AR-mediated activity in the follicle and are the focus of most studies in this area of investigation. The AR-mediated role for androgens in folliculogenesis and its mechanism of action are still being characterized. Nonhuman primates [19] and women [20, 21] exposed to high serum androgens develop large ovaries with increased numbers of antral follicles. Both testosterone and DHT promote in vitro follicle growth in mice [22, 23] and DHT treatment enhances ovulation rate in pigs [24]. Many of the differentiative actions of FSH on granulosa cells are augmented by AR agonists and include cholesterol metabolism, progesterone secretion, expression of steroidogenic enzymes, and induction of aromatase activity (reviewed in [25]). The mechanism involves modulation of cAMP within granulosa cells at both pre- and post-cAMP sites [25] and presumably involves the regulation of androgen-responsive genes. Although definitive proof of direct AR transcriptional regulation is still lacking, the FSH receptor (FSHR) may be one such ovarian target gene [24, 26, 27]. However, androgens may also act via other systems that impact on FSH activity. In addition to FSHR, androgen treatment also enhances insulin-like growth factor (IGF)-1 and IGF receptor mRNA expression in granulosa cells [28] and oocytes [29] of rhesus monkeys. Enhancement of FSH-mediated activity by IGF-I is well documented and also occurs at both pre- and post-cAMP sites [30, 31]. However, the effect of androgens on IGF-I mediated activity, alone or in combination with FSH, has not been explored in cultured granulosa cells.
In the current study, we have used a prepubertal pig model to study the effects of androgen on granulosa cell responses to FSH and IGF-I. As the interaction of these two mitogens changes in the presence of oocyte-secreted factors [32], we included in our investigation the two subpopulations of granulosa cells that emerge upon antrum formation in the follicle: mural granulosa cells (MGCs) that comprise the follicle wall and cumulus cells, which are intimately connected to the oocyte via gap junctions forming a cumulus-oocyte complex (COC). These two granulosa cell subtypes are anatomically and functionally distinct (reviewed by Eppig et al. [33]) due to varying proximities to the oocyte and the vascularized thecal layer. Both subtypes display strong immunological reactivity for AR in pigs [34]. We anticipated differential androgen responses between these two granulosa cell subtypes and we report, to our knowledge, for the first time, that proximity of the oocyte corresponds to increased androgenic activity that manifests as altered granulosa cell responses to mitogenic stimulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise noted, all chemical reagents were purchased from Sigma (St. Louis, MO). Prepubertal pig ovaries were collected from a local abattoir and transported to the laboratory in warm saline supplemented with penicillin G (100 U/ml) and streptomycin sulphate (100 mg/ml). Time from ovary excision to follicle aspiration was approximately 1–2 h. Follicular cells were aspirated from antral follicles using a Precision Glide needle (0.9 mm x 25 mm) inserted in a 10-ml Vacutainer tube (both from Becton Dickinson, Meylan, France) under vacuum; ovaries with corpora lutea and follicles that were opaque or hemorrhagic were excluded. Follicular aspirates were kept at 37°C and allowed to sediment for 5 min. The sediment was transferred to nontreated Petri dishes containing warm 25 mM HEPES-buffered tissue culture medium-199 (H-TCM; ICN, Costa Mesa, CA) supplemented with 2 mM sodium pyruvate, 100 U/ml penicillin G, streptomycin sulphate (100 mg/ml), and polyvinyl alcohol (0.3 mg/ ml). With the aid of a dissecting microscope, all naked oocytes and COCs were removed from the aspirates and placed into fresh H-TCM (as described above). From these, COCs with good structural integrity were further selected and manually washed twice in H-TCM and twice in bicarbonate-buffered TCM-199 (B-TCM, supplemented as per H-TCM; ICN) before culturing. Debris was removed from the remaining aspirate, which was then collected into a centrifuge tube and allowed to sediment by unit gravity for 1 min. This sediment contained large aggregates of mural granulosa cells (MGCs) and was discarded. The remaining aspirate was allowed to sediment a further 30 min, creating a pellet of aggregated MGCs of a more uniform size range. The supernatant, containing mostly single cells, was discarded and the pellet was washed twice in H-TCM and twice in B-TCM, with centrifugation between washes. An aliquot of resuspended cells was manually dissociated using a 1-ml syringe with a 23-gauge needle and counted with a hemocytometer to determine cell density.
Follicle Cell Culture
Mural granulosa cells were cultured as aggregates in 96-well flat-bottom plates (Falcon, Franklin Lakes, NJ) at a density of 106 cells/ml in a culture volume of 250 µl. Cumulus-oocyte complexes (10/well) were cultured in 96-well round-bottom plates (Falcon) in a culture volume of 125 µl. Culture conditions were serum-free B-TCM, supplemented as above, with combinations of the following hormone treatments: 50 mIU/ml recombinant human FSH (rhFSH; Puregon, N.V. Organon, Oss, Netherlands), 50 ng/ml recombinant human IGF-I (rhIGF-I; Gropep, Adelaide, Australia), 0.5 µM DHT (Sigma), and 5 µM hydroxyflutamide (OHF; Sigma). Cells were cultured with treatments in an atmosphere of 38.5°C, 96% humidity, 5% CO2 in air for 18 h followed by a 6-h pulse of 0.8 µCi tritiated thymidine ([H3]-thymidine; ICN) in the same conditions. At 24 h, a fraction of the culture media was removed and frozen (–20°C) for steroid analysis, and plates were kept at 4°C until cell harvest.
Effects of Androgens on Proliferative and Steroidogenic Responses to FSH and IGF-I in MGCs and COCs
To test the responses of MGCs and COCs to an AR agonist, we used the nonaromatizable DHT at 0.5 µM, a concentration that approximates the level of this steroid in follicular fluid. In separate experiments, cells were obtained from either 3- to 5-mm or 1- to 3-mm antral follicles. Cell numbers were not restrictive for MGCs and all treatments were done on one plate in triplicate or quadruplicate, repeated in five experiments. As COCs were limited in number, smaller treatment groups were employed with 2–4 duplicates per treatment. Each experimental condition was repeated in a minimum of 4 experiments.
Effects of Androgen Receptor Antagonist on Proliferative and Steroidogenic Responses of MGCs and COCs to FSH, IGF-I, and Androgens
Because most steroids, including DHT, have documented nongenomic effects that are independent of their cognate receptor [35], the androgen receptor antagonist, OHF, was used to test the specificity of the observed androgen responses. MGCs or COCs were cultured under conditions selected on the basis of significant androgen effects that were observed in the above experiments. Hydroxyflutamide was employed at 5 µM, a 10-fold excess above that of DHT. Each experiment was repeated a minimum of four times with triplicate or quadruplicate treatments in MGCs and 2– 3 duplicate treatments in COCs.
Time-Course of Proliferative Responses to FSH, IGF-I, and Androgens
MGCs from 1- to 3-mm antral follicles were cultured as described above, but for each experiment, four separate culture plates were established, corresponding to 6-hourly termination points. Tritiated thymidine was added to cultures 6 h before termination of culture for each time point. The experiment was repeated three times with 3–6 duplicated treatments per plate.
Measurement of DNA Synthesis and Progesterone Secretion
Following culture, a fraction of media was removed and assayed for progesterone using an RIA kit (Diagnostic System Laboratories, Webster, TX) in accordance with the manufacturer's instructions. The kit has a sensitivity of 0.25 pmol/ml, an intraassay coefficient of 8.4%, and an interassay coefficient of 12%. Incorporation of [3H]-thymidine (ICN) was measured in cells as an indication of the degree of cellular DNA synthesis and potential proliferation. Cells were harvested onto a filter mat using a Tomtec Harvester 96 (Tomtec, Hamden, CT), which removes unincorporated isotope in the process. Filter mats were then saturated with scintillation fluid, and emission of beta particles by [3H]-thymidine was detected with a Wallac microbeta counter (Fisions, Leies, UK). In independent experiments, incorporation of [3H]-thymidine into DNA was confirmed using methods similar to those described by Yong et al. (data not shown) [36].
Data Analysis
Statistical analyses were performed with SAS software (SAS Institute, Cary, NC) using three-way ANOVAs on the log response of raw data, with blocking on experiment. Where significant effects were seen, post hoc t-tests were used for comparison of adjusted means for those significant effects. For clarity of presentation, raw data were converted to relative data for each replicate experiment, in which the mean value for the control treatment with no hormone was designated as 1. Graphs therefore represent the mean values (±SEM) of the summation of relative data for a group of replicate experiments. Statistical significance was set at P < 0.05, except where otherwise noted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of Androgen on Proliferative and Steroidogenic Responses of MGCs and COCs to FSH and IGF-I
Three- to five-millimeter antral follicles Figure 1 summarizes the effects of FSH, IGF-I, and DHT alone and in combinations on DNA synthesis and progesterone secretion in MGCs and COCs from 3- to 5-mm antral follicles. Both FSH and IGF-I significantly increased DNA synthesis in MGCs, and the responses to these agents in combination were approximately additive (Fig. 1A). Similarly, both FSH and IGF-I stimulated progesterone secretion in MGCs, with the effect of FSH considerably greater than that of IGF-I. The effect of combined treatment with FSH plus IGF-I on progesterone secretion was a greater than additive response that tended toward synergism (Fig. 1C).
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Proliferative effects of FSH and IGF-I were also seen in COCs (Fig. 1B), although the magnitudes of stimulation by both agents were considerably greater than observed in MGCs, and the response to IGF-I was substantially greater (20-fold stimulation) than the response to FSH (8-fold stimulation). A more striking difference between the two granulosa cell subtypes was seen in response to combined treatment with FSH and IGF-I, in which FSH completely obliterated the proliferative response to IGF-I. The pattern of steroidogenic responses of COCs to FSH and IGF-I alone and in combination were similar to those observed in the MGCs, although the magnitude of progesterone secretion was considerably less in the COCs.
The nonaromatizable androgen, DHT, had a significant main effect on DNA synthesis in MGCs (three-way ANOVA; P = 0.04) but there were no significant interactions between androgen and FSH or IGF-I. Progesterone secretion in MGCs was globally unaffected by DHT (Fig. 1C). To exclude the possibility of an inappropriate dose of androgen, the same treatments were repeated in combination with a range of DHT concentrations from 1 nM to 1 µM, with no significant effect on FSH and/or IFG-1-stimulated DNA synthesis or progesterone secretion (data not shown). In COCs, the three-way interaction of FSH, IGF-I, and DHT was close to significance (P = 0.06), and post hoc tests assuming significance at this level show augmentation of FSH-stimulated proliferation by DHT (P < 0.0001) (Fig. 1B). Interestingly, although DHT did not alter proliferative responses of COCs to IGF-I alone, in the presence of both mitogens, DHT reverses to a small but significant degree (P = 0.02) the inhibitory effect of FSH on IGF-I-stimulated proliferation. The steroidogenic response of COCs to FSH was inhibited 2-fold by DHT (P < 0.0001) (Fig. 1D), independent of the presence of IGF-I. Progesterone secretion from COCs under control conditions and DHT alone was undetectable and therefore arbitrarily given the value of the lowest assay standard in the graph.
One- to three-millimeter antral follicles Figure 2 summarizes the effects of FSH, IGF-I, and DHT alone and in combinations on DNA synthesis and progesterone secretion in MGCs and COCs from 1- to 3-mm follicles. The pattern and degree of mitogen-stimulated DNA synthesis in MGCs from these follicles (Fig. 2A) differed from that seen in MGCs from follicles 3–5 mm in diameter (Fig. 1A). The relative degree of FSH stimulation above control values was approximately 10-fold in small follicles compared with approximately 2-fold in the larger ones, resulting in a 6-fold higher uptake of [3H]-thymidine in cells from smaller follicles. While in relative terms, IGF-I alone was a less potent mitogen than FSH in cells from small follicles, in terms of total uptake of [3H]-thymidine, IGF-I induced similar effects in cells from both follicle sizes. Another consistent feature of responses of MGCs from both follicle sizes was the enhanced effect of combined mitogen over either mitogen alone, although this effect was approximately 20-fold in small follicles as compared with approximately 4-fold in larger follicles due to the greater mitogenicity of FSH in the former. Progesterone secretion from MGCs of 1- to 3-mm follicles (Fig. 2C) also followed similar patterns of response to FSH and IGF-I as observed in MGCs from 3- to 5-mm follicles, although cells from smaller follicles secreted a greater amount than cells from larger follicles under all conditions.
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The effects of DHT were more ubiquitous in MGCs from 1- to 3-mm follicles as compared with MGCs from 3- to 5-mm follicles; there was a highly significant main effect of DHT on both DNA synthesis (three-way ANOVA; P < 0.001) and progesterone secretion (three-way ANOVA; P < 0.0001) in cells from the smaller follicles. In terms of DNA synthesis, there was also a trend toward a three-way interaction between DHT, FSH, and IGF-I (P = 0.08) in MGCs from smaller follicles that was not apparent in MGCs from larger follicles. Post hoc tests assuming this interaction was significant show an enhancement of IGF-I-stimulated DNA synthesis by DHT (P = 0.0003) (Fig. 2A). The effects of DHT on steroidogenic responses of MGCs from 1- to 3-mm follicles were inconsistent: in some assays, DHT inhibited FSH-stimulated progesterone secretion and in others it did not, leading to comparisons that were not statistically significant (Fig. 2C). However, the main effect of DHT on progesterone secretion in cells from smaller follicles was highly significant (three-way ANOVA; P < 0.0001).
Mitogen-stimulated DNA synthesis (Fig. 2B) and progesterone secretion (Fig. 2D) characteristic of COCs from 1- to 3-mm follicles followed similar patterns of stimulation as observed in COCs from larger follicles (Fig. 1, B and D). In terms of DNA synthesis, baseline values for control and FSH-stimulated COCs were approximately 2-fold greater in COCs from 3- to 5-mm follicles, but the amount of progesterone secretion was nearly identical between COCs of both follicle sizes. Stimulation by IGF-I and combined mitogen was also remarkably similar in relative and absolute terms between COCs from both follicle sizes in assessment of both DNA synthesis and progesterone secretion.
In COCs from 1- to 3-mm follicles, there was a significant three-way interaction between DHT, FSH, and IGF-I (P = 0.05) in stimulating DNA synthesis, whereby DHT augmented IGF-I-stimulated DNA synthesis to approximately 50-fold over controls (P < 0.0001; Fig. 2B). This feature was absent in COCs from 3- to 5-mm follicles, where the androgen effect was only evident in the presence of FSH. Dihydrotestosterone also significantly enhanced FSH-stimulated DNA synthesis in COCs from 1- to 3-mm follicles (P < 0.01) and tended to increase DNA synthesis on its own (P = 0.07) (Fig. 2B). In addition, as observed in COCs from 3- to 5-mm follicles, DHT inhibited progesterone secretion by approximately 2-fold in the presence of FSH, independent of the presence of IGF-I in COCs from 1- to 3-mm follicles (P < 0.0001; Fig. 2D).
Effect of an Androgen Receptor Antagonist, Hydroxyflutamide, on Granulosa Cell Responses to DHT
The AR antagonist OHF was able to abolish the stimulatory effects of DHT on both FSH-stimulated DNA synthesis in COCs from 3- to 5-mm follicles (Fig. 3A) and IGF-I-stimulated DNA synthesis in COCs from 1- to 3-mm follicles (Fig. 4B). However, DHT and OHF had significant two-way interactions with IGF-I (P = 0.05 and P = 0.001, respectively), independently stimulating DNA synthesis in MGC from 1- to 3-mm follicles and having a greater effect when combined than either alone (Fig. 4A). Likewise, OHF had agonistic effects similar to DHT in the inhibition of FSH-stimulated progesterone secretion in COCs from 3- to 5-mm follicles (Fig. 3B; two significant two-way interactions: FSH x DHT, P = 0.02; FSH x OHF, P = 0.001) and was more potent than DHT in suppressing progesterone secretion in the absence of FSH (significant two-way interaction between DHT x OHF, P < 0.001). As COCs from small antral follicles do not secrete measurable quantities of progesterone under control or IGF-I conditions, the effects of OHF were not examined.
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Time-Course of Proliferative Responses of MGC to FSH, IGF-I, and Androgens
Six-hourly time points were used to observe the progressive effects of mitogens and androgens on DNA synthesis in MGC from small antral follicles over a 24-h period (Table 1). Under control conditions, MGCs dramatically lost their ability to incorporate [3H]-thymidine after 6 h in culture, exhibiting a 50-fold decrease in uptake by 24 h. As expected, addition of FSH and IGF-I, alone or in combination, maintained a higher level of [3H]-thymidine incorporation in these cells as compared with control conditions at each time point. Interestingly, at both 6 and 12 h, MGCs exhibited a response to mitogen that is similar to that exhibited by COCs, whereby IGF-I was a more potent stimulus than FSH, and FSH tended to inhibit IGF-I stimulation when both mitogens were present (P < 0.001 in all instances). During this first half of the culture period, DHT had no effect on [3H]-thymidine incorporation under any condition. The second 12-h culture period was distinct from the first 12-h period in three ways: 1) the mitogenic effect of FSH exceeded the effect of IGF-I at both 18 (P < 0.001) and 24 h (P < 0.0001); 2) the two mitogens combined had an additive effect larger than each mitogen alone, and 3) responses to DHT emerged (significant three-way interaction between DHT, FSH, and IGF-I, P < 0.05 at both 18 and 24 h). The effect of DHT was most notable in combination with IGF-I. At 18 h, DHT was able to enhance the mitogenic stimulation of IGF-I, and at 24 h, there was a two-fold increase in [3H]-thymidine incorporation over IGF-I alone (P < 0.0001 in both instances). In this series of experiments, DHT also tended to enhance FSH-stimulated DNA synthesis (P = 0.06).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alone, DHT exerted a main effect on proliferation in both granulosa cell subtypes from both follicle sizes; however, this effect was strongest in granulosa cells from 1- to 3-mm follicles, particularly in those cells complexed to the oocyte. Although we did not examine preantral granulosa cells in this study, these cells have intense expression of AR protein in most species, including the pig [39], and it is feasible that androgen effects on proliferation could be stronger in preantral cells. Cultured preantral mouse follicles respond directly to androgens by increasing follicle diameter and DNA synthesis [23]. However, previous to our study, a direct in vitro effect of androgen alone on proliferation of granulosa cells had not been demonstrated for nonmurine species, although the presence of AR has been positively associated with the cell-cycle-related nuclear antigen Ki-67 [40].
Stimulatory effects of androgen on the proliferation of granulosa cells were most pronounced in the presence of a singular mitogen, and the specificity of that mitogen was determined by follicle size. Thus, in granulosa cells from small antral follicles, DHT had the most significant and consistent effect in the presence of IGF-I, whereas in granulosa cells from medium-sized antral follicles, this effect was lacking, and androgen either had no effect (MGCs) or enhanced FSH-stimulated mitogenesis in the presence of the oocyte (COCs). Androgen also induced a small increase in FSH-stimulated proliferation in MGC from small antral follicles, an effect preceded in a time course trial by enhancement of IGF-I-stimulated mitogenesis. Studies by Vendola et al. using androgen-treated monkeys as a model show marked increases in mRNA for IGF-I and IGF receptor [28, 29], and FSHR [19] in similar-sized follicles. Our data support the hypothesis introduced by these authors that androgens may be acting in part through mediation of IGF-I activity, at least in small antral follicles. Whether this occurs through induction of FSHR and is responsible for the effect of androgen on FSH-mediated mitogenesis remains to be determined. However, enhancement of IGF-I activity could potentially explain the effect of DHT alone on proliferation of granulosa cells in our study and of androgens on in vitro mouse follicle growth [22, 23].
Numerous studies have demonstrated the enhancement of differentiative actions of FSH by androgens, but few have examined their effect on the mitogenic capacity of this gonadotrophin. Bley et al. [41] demonstrated enhanced FSH-stimulated DNA synthesis by DHT under serum-free conditions in cultured rat granulosa cells, but only after 48 h. At 24 h, the steroid had no effect, consistent with our data for MGC from 3- to 5-mm antral follicles. However, we observed this mitogenic response in COCs from the same follicles, coincident with a suppression of FSH-stimulated progesterone secretion that was not observed in the MGCs. Only MGCs from small antral follicles, that may not have fully differentiated from the more primitive preantral granulosa cells, exhibited responses to DHT that were similar to those observed in COCs. An inverse relationship between proliferation and progesterone secretion has been widely demonstrated in granulosa cells, whereby factors that stimulate one activity also suppress the other [32, 38]. This pertains to androgens, which have been shown to suppress granulosa cell DNA synthesis [42, 43] and enhance granulosa cell steroidogenesis [5, 44], the opposite to our current findings. In the earlier studies, granulosa cells were cultured under conditions that promote a more luteinized granulosa cell phenotype. Collectively, these observations suggest that the differentiative differences incurred on granulosa cells by oocyte-secreted factor(s) influence the ability of androgens to alter both the mitogenic and steroidogenic actions of FSH and IGF-I in antral follicles. Current research in our laboratory, in which denuded oocytes are cocultured with MGCs, is aimed at further characterizing this phenomenon [45].
One means by which oocytes may affect androgenic responses is via regulation of AR expression. It has been demonstrated in rats [46], pigs [39], and monkeys [40, 47] that MGC from small follicles possess more AR protein and mRNA than larger follicles. Hillier et al. [25] suggest that this reduction in AR is necessary for the transition to preovulatory growth, a suggestion further supported by recent data from Cheng et al. [48] in which high expression of AR in the granulosa cells of ERß-deficient mice was observed together with a block in progressive antral follicle growth. Our data provide evidence that one physiological consequence of a reduction in AR expression in MGCs is loss of the ability of androgen to stimulate DNA synthesis and suppress progesterone secretion. Furthermore, our results are in accordance with those of Garrett and Guthrie [39], that these events occur when antral follicles reach 3 mm in diameter in the pig. Although quantitative comparisons of the expression of AR between cumulus and mural granulosa cells are lacking, two studies anecdotally observe that AR expression diminishes in a centripetal fashion toward the oocyte in rats [46] and pigs [34]. We demonstrate that androgens continue to enhance FSH-stimulated mitogenesis and suppress FSH-stimulated progesterone secretion in COCs from 3- to 5-mm follicles, suggesting that modulation of androgen activity by the oocyte may be a means of maintaining a distinct phenotype.
When studying androgen activity, it is common to use DHT as a ligand, the activity of which is implicitly linked with the AR, and effects induced by this androgen are generally presumed to follow classic genomic steroid receptor pathways involving modulation of the transcriptional activity of target genes. Using the AR antagonist hydroxyflutamide (OHF), we found that enhancement of mitogen-stimulated proliferation by DHT generally followed a pattern of classic steroid activity but suppression of FSH-stimulated progesterone secretion by DHT and OHF was indicative of nonclassic steroid activity. Antiandrogens have been shown to inhibit steroidogenic enzyme activity in rat testicular tissue [49] and to have a differential effect on FSH- and cAMP-induced steroidogenesis in rat granulosa cells [44]. The ability of OHF to antagonize certain androgenic effects but agonize others may be due to its unique interaction with the AR: it can bind the AR and induce a conformational change that can have androgenic effects on cytoplasmic signal transduction pathways [50] but is unable to induce the conformational change necessary for DNA binding and gene regulation [51]. The finding that OHF can be agonistic in the presence of DHT and IGF-I in stimulating proliferation of MGCs from small follicle, but antagonistic to proliferation under the same conditions in cumulus granulosa cells from the same follicles is difficult to explain, but again highlights the influence of the oocyte. Zhu et al. [50] have reported differential effects of OHF on proliferation of human breast cancer cells and provide evidence that both genomic and nongenomic mechanisms are involved. Our data suggests that AR-mediated influences on steroidogenesis and mitogenesis in granulosa cells are more complex than the regulation of gene transcription, although the specific mechanisms at play remain unresolved.
In summary, we demonstrate that androgens mainly enhance mitogen-stimulated proliferation and suppress FSH-induced differentiation in immature granulosa cells of the pig and that this activity is promoted by proximity to the oocyte. As the antrum enlarges and two granulosa cell subtypes become established, differential androgen activity emerges whereby those cells more distant from the oocyte become less responsive to androgen. This change in androgen activity appears to correlate with a reduction in AR expression and subsequent luteinization of the granulosa cells. Androgenic activity in immature granulosa cells is complex and possibly mediated by classic and nonclassic steroid mechanisms; both appear to be influenced by the oocyte. Our data add to the growing compendium of evidence that the oocyte orchestrates developmental events in folliculogenesis. We suggest that control of androgenic activity can be added to its repertoire and may be one means by which the oocyte abrogates differentiation of granulosa cells toward a luteinized phenotype.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Theresa E. Hickey, Department of Obstetrics and Gynaecology, The University of Adelaide, The Queen Elizabeth Hospital, 1st Floor Maternity Building, Woodville Road, Woodville, SA 5011, Australia. FAX: 61 8 8222 7521; theresa.hickey{at}adelaide.edu.au
Received: 12 December 2003.
First decision: 22 December 2003.
Accepted: 13 February 2004.
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