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Regular Article |
a Departments of Obstetrics and Gynecology, Leland J. and Dorothy H. Olson Center for Women's Health,
b Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
ABSTRACT
Epidermal growth factor (EGF) is mitogenic to preantral follicles, and transforming growth factor ß (TGFß) influences ovarian cell functions in a variety of species. Although an interaction of these ligands during preantral folliculogenesis is likely, whether EGF influences TGFß action on preantral follicles by modulating TGFß receptor (TßR) gene transcription and translation is not known. To determine whether EGF influenced TßR mRNA and protein levels in granulosa cells during preantral folliculogenesis, hamster preantral follicles at stages 1–6 were cultured in the absence or presence of EGF and follicular TßR mRNA, and protein levels were monitored by semiquantitative reverse transcription polymerase chain reaction and immunoblotting, respectively. Both TßR type I (TßRI) and TßR type II (TßRII) mRNA and protein were present in preantral follicles, and their expression was up-regulated by EGF in a stage-dependent manner. However, EGF effect on the expression of TßRI and TßRII was differential. In contrast to TßRI, EGF-stimulation of follicular TßRII mRNA expression was evident from stages 1 and 2 onwards, and more than twofold induction was noted for stages 4–6. Moreover, significant increases in thecal TßR mRNA levels were noted for stage 6 follicles. Follicles at smaller stages appeared to be more sensitive to EGF than were larger preantral follicles. Despite an increase in the cytosolic form of TßRI protein for most of the stages and TßRII protein for follicles at stages 4 and 5, EGF-stimulation of the membrane-associated form of the receptor was restricted to follicles at stage 6. Functionally, TGFß1 attenuated EGF-induced DNA synthesis for follicles at stages 1–3 and 6 without affecting EGF-induced progesterone production for most of the stages. Administration of
-amanitin resulted in a significant reduction of EGF-induction of TßR mRNA levels, suggesting that increased receptor protein levels were a consequence of mRNA synthesis. These results indicate that an interaction between EGF and TGFß forms an important regulatory mechanism for preantral folliculogenesis. The effect of EGF on TßRI and TßRII gene transcription and translation are differential, and follicular response to EGF depends on the developmental status of the follicles.
follicle, follicular development, gene regulation, growth factors, ovary
INTRODUCTION
Among the mitogenic factors, epidermal growth factor (EGF), transforming growth factor , insulin-like growth factor I, and their binding proteins and cognate receptors have been detected in ovarian cells from a variety of species [1–4]. Likewise, transforming growth factor ß (TGFß) and its cognate receptors have been identified in ovaries of many species [1–3, 5]. On the contrary, only EGF [6], TGFß [7], and TGFß receptor (TßR) [8] have so far been detected in hamster ovarian follicles [6–8]. EGF induces granulosa cell proliferation, and TGFß is one of the most potent cell differentiation factors [9]. In the hamster, EGF stimulates granulosa cell proliferation, whereas TGFß inhibits EGF action on follicular cells [10]. TGFß has been shown to influence granulosa cell functions in a variety of species [1]. In rat granulosa cell culture, TGFß augments FSH-stimulated LH receptor induction and progesterone production [11]. Because of the opposing effects of EGF and TGFß on the hamster granulosa cells, the timing of the expression of these two factors may be critical in determining the success of follicle development. The population of granulosa cells in follicles is asynchronous; hence, proliferative cells are present in preantral and small antral follicles at all stages, and follicular cells undergo gradual differentiation, such as the acquisition of complete steroidogenic potential, induction of LH receptors, and synthesis of follicle specific proteins, throughout folliculogenesis [1]. Therefore, it is logical to assume that the proliferative and differentiation phases should alternate for timely development of ovulatory follicles. In human preantral follicles, EGF enhanced TßR expression [12]. Of the TßRs, type I (TßRI) and type II (TßRII) are involved in mediating TGFß action [13], and the expression of both types of receptor is essential for the biological action of TGFß [14, 15]. The biological action of TGFß on hamster and rat follicular cells [10, 16, 17] and the presence of TßR protein [8] and mRNA [18] in hamster ovarian cells have been documented. Hamster granulosa cells possess two forms of TßRII: a membrane-bound form that recognizes the ligand and an abundant cytoplasmic form that shows limited interaction with the ligand [8]. Similarly, EGF has been localized in hamster granulosa cells [6], and EGF-induced proliferation of granulosa cells has been documented for many species, including the hamster [10, 19, 20]. The presence of EGF receptor in ovarian cells has also been documented [21–25]. Based on these lines of evidence, it is likely that EGF and TGFß interact to regulate each other in their actions on granulosa cells. However, the mechanisms of EGF modulation of TGFß action on granulosa cells remain unclear. The objective of the present study was to determine whether modulation of TßR mRNA and protein expression during hamster preantral folliculogenesis was one of the potential mechanisms whereby EGF could influence TGFß effect.
MATERIALS AND METHODS
Materials
All oligodeoxynucleotide primers were synthesized by Genosys Biotechnologies (The Woodlands, TX). Reverse transcription polymerase chain reaction (RT-PCR) chemicals were obtained from Life Technologies (Rockville, MD), Boehringer/Roche Molecular Biochemicals (Mannheim, Germany), or Amersham/Pharmacia (Piscataway, NJ). Zeta probe nucleic acid transfer membrane was from Bio-Rad (Hercules, CA). Tri-Reagent for RNA extraction was from MRC (Cincinnati, OH) and [
-32P]ATP (specific activity, 7000 Ci/mmol) was from ICN Radiochemicals (Costa Mesa, CA). Polyclonal antibodies to TßRI and TßRII were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Protein A/G agarose and the chemiluminescence kit were obtained from Pierce Chemical Co. (Rockford, IL). Dulbecco modified Eagle medium (DMEM), collagenase, DNase, detergents, and other analytical grade chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Adult cyclic golden hamsters were purchased from SASCO (Madison, WI).
Female golden hamsters (90–100 g) were maintained in a temperature- and light (14L:10D)-controlled environment according to NIH and Institutional Animal Care and Use Committee (IACUC) guidelines. The use of hamsters was approved by the IACUC. Animals with at least three consecutive estrous cycles were used for all experiments.
Experiment 1: Effect of EGF on Follicular TßR mRNA Levels
Preantral follicles at stages 1+2 through 5 (S1+2–S5; S1–S4 = one to four layers of granulosa cells, S5 = five or six layers of granulosa cells and no thecal layer) and stage 6 (S6 = seven or eight layers of granulosa cells and developing thecal layers) were collected on Day 4 at 0900 h by enzymatic dissociation and dissection [26], respectively, and cultured in DMEM containing 1% TS+ (0.1 µg/ml transferrin, 0.1 µg/ml selenium), 0.1 µg/ml insulin, and 40 ng/ml hydrocortisone [10] for 24 h at 37°C under 5% CO2 in air. The effect of 5, 10, 25, 50, or 100 ng/ml EGF was tested, and the time course of action of an optimal dose of EGF was determined by culturing follicles for 6, 12, 24, or 48 h. Follicles were retrieved for RNA isolation and TßR mRNA analysis.
TßRI, TßRII, and S4 mRNA levels were determined by a semi-quantitative RT-PCR followed by Southern hybridization and phosphorimaging quantitation of the cDNA signal essentially as previously described [18]. The data were expressed as fold change relative to untreated follicles to normalize for the variation in receptor mRNA levels in follicles at different stages.
Experiment 2: Effect of EGF on Follicular TßR Protein Levels
Follicles at S1+2–S6 were isolated from ovaries of a proestrous hamster at 0900 h and cultured as described in experiment 1. An optimal dose of EGF (50 ng/ml) [20] was added at the onset of culture, and follicles were retrieved after 24 h and processed for the analysis of both cytosolic and membrane-associated forms of TßRI and TßRII as previously described [8] with minor modifications. Cultured control and EGF-treated follicles were placed in 50 µl of ice-cold 10 mM Tris-glycerol buffer, pH 7.0, containing 10% glycerol, 0.25 M sucrose, 200 µM sodium vanadate, and 100 µl protease inhibitor cocktail (Sigma). The follicles were sonicated twice at 20 W for 5 sec each on ice. Homogenates were then centrifuged at 100 000 x g for 30 min at 4°C, the supernatant was separated from the pellet, and the pellet was resuspended in 1x RIPA (10 mM PBS, pH 7.0, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) for 30 min on ice and centrifuged again at 26 000 x g for 20 min at 4°C for membrane supernatant. The protein content of the samples was determined by a Micro BCA protein assay kit (Pierce), and equal amounts of cytosolic or membrane protein for each stage was clarified with 1 µg of nonimmune rabbit IgG and 10 µl of protein A/G agarose (Pierce) either in 500 µl of Tris-glycerol buffer (for the cytosol) or 1x RIPA (for the membrane) for 4 h at 4°C. The samples were mixed overnight with 1 µg of anti-TßRI or anti-TßRII IgG and 10 µl of protein A/G agarose [8]. The agarose-antibody-antigen complex was washed three times with 1x RIPA at 2000 x g, resuspended in 20 µl of 2x SDS-PAGE buffer, fractionated in a 7.5% polyacrylamide gel, and electrotransferred to nitrocellulose membrane (Optitran, ISC BioExpress, Kaysville, UT). The blot was probed with the same antibodies that were used for immunoprecipitation, and the signal was generated using a chemiluminescence system and chemiluminography as described previously [8]. The signal was quantified using a laser densitometer.
Experiment 3: Effect of EGF and TGFß Interaction on Follicular DNA Synthesis and Progesterone Production
Follicles at S1+2–S6 were isolated from ovaries of proestrous hamsters at 0900 h and were cultured for a total period of 48 h with or without 50 ng/ml EGF. After the initial 24 h of culture, one group of control and EGF-exposed follicles received 5 ng/ml of TGFß1, and the culture was continued for an additional 24 h. To evaluate DNA synthesis, 5 µCi/ml [3H]thymidine was added to all cultures 24 h before termination. The medium from each 24-h culture was saved to measure progesterone. Follicles were processed for quantifying DNA by fluorometry and [3H]thymidine incorporation by scintillation spectrometry as previously described [27, 28]. The results of [3H]thymidine uptake were expressed as counts per minute (cpm) per nanogram of DNA per 24 h. Progesterone accumulation in the medium was assayed by specific RIAs as described previously [19, 29]. All samples were assayed at the same time to avoid interassay variation, and the intra-assay variation was 7%. The results were expressed as picograms of progesterone per follicle for 24 h.
Experiment 4: Effect of -Amanitin on Follicular TßRI and TßRII mRNA Levels
To determine whether the increase in follicular TßR mRNA levels following EGF exposure was due to increase in receptor mRNA synthesis, follicles at S1+2–S6 were cultured for 12 h in the absence or presence of 50 ng/ml of EGF and 25 µg/ml of -amanitin [30] to block any transcriptional activity. The 12-h exposure time was based on the results of experiment 1, when EGF effect was evident for most of the stages. Follicular RNA was used to determine the levels of TßRI and TßRII mRNA with respect to S4 mRNA as described earlier. Incorporation of [3H]leucine in follicular protein was determined by precipitating protein from follicular homogenates using 10% ice-cold trichloroacetic acid (TCA), rinsing the pellet twice with 5% TCA, and finally dissolving the pellet in 1 M NH4OH and counting the sample in a Beckman scintillation spectrometer. Protein concentration of the homogenate was measured using a Micro BCA protein assay kit (Pierce). The data were expressed as counts per minute of [3H]leucine incorporated per milligram of protein per 12 h. The effect of -amanitin was not due to generalized cell death as determined by culturing S3 follicles in the absence or presence of -amanitin for 12 h and in the presence of 10 µCi/ml [3H]leucine (specific activity, 162 Ci/mmol), which was added 6 h before culture termination.
Data Analysis
All experiments were repeated at least three times using a pool of follicles collected on three different occasions to obtain sample sizes of 3. Values (DLU [digital light unit] target/DLU S4) for untreated and treated groups were analyzed by a two-way ANOVA and a Fisher exact test using Jandel Sigmastat Statistical software Version 2.0 (Statistical Solutions, Saugus, MA) to determine the significance of any differences (P < 0.05). Because of the variation in basal levels of receptor mRNA across stages and for clarity, receptor mRNA expression for treated groups was presented as fold change relative to controls. Further, each bar in Figures 1 and 2 represents the mean ± SEM of three "fold difference" values. All samples were processed at one time to avoid interassay variation, and the intra-assay variation was around 5%, which was calculated from multiple tubes of internal positive controls.
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RESULTS
Effect of EGF on Follicular TßR mRNA Levels
Both TßRI and TßRII mRNAs were present in granulosa cells of S1+2–S6 and in thecal cells of S6. EGF differentially influenced follicular TßRI and TßRII mRNA levels in a stage- and time-dependent manner (Figs. 1 and 2). Whereas increases in TßRI mRNA levels were observed from S3 onwards (Fig. 1a), significant increase in TßRII mRNA expression was noted for S1+2–S6 (Fig. 1b). Further, significant (P < 0.05) stage-specific variation in follicular sensitivity to EGF was evident (Fig. 1, a and b). More than twofold induction of TßRI mRNA occurred in granulosa cells in response to 25 ng/ml EGF for follicles at S3–S6 (Fig. 1a). Only 50 ng/ml EGF was effective in inducing thecal TßRI mRNA levels for S6 (Fig. 1a). In contrast to TßRI, consistent EGF effect on follicular TßRII mRNA levels was more pronounced for S4–S6; however, S1+2 and S3 responded significantly to certain dosages of EGF (Fig. 1b). Thecal TßRII mRNA levels for S6 increased as much as sixfold in response to 50 ng/ml EGF (Fig. 1b). Likewise, maximum response to EGF for TßRI mRNA was evident by 6 h for all but S6, for which significant (P < 0.05) increase was noted by 3 h followed by a second rise by 24 h (Fig. 2a). However, no further increase was noted after 48 h for any stage (Fig. 2a). In fact, a decrease was evident for all but S5 follicles (Fig. 2a). Whereas EGF could still maintain the high steady-state levels of TßRI mRNA in follicles at S4–S6 beyond 12 h, no response was evident for S1+2 and S3 (Fig. 2a). On the contrary, follicular response to EGF stimulation of TßRII mRNA levels differed markedly among stages (Fig. 2b). Whereas maximum response was evident by 6 h for S1+2–S4, significant increase was observed by 12 h for S5 and by 3 h for S6 follicles (Fig. 2b). Similar to observations for TßRI mRNA, a second rise in TßRII mRNA levels was evident for S6 follicles by 48 h (Fig. 2b). Moreover, in contrast to TßRI mRNA expression, the steady-state level of TßRII mRNA was maintained for most of the stages through 48 h (Fig. 2b).
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Effect of EGF on Follicular TßR Protein Levels
EGF significantly stimulated follicular protein synthesis after 24 h of incubation (control: 80 ± 5 cpm-1 µg protein-1 6 h; EGF: 145 ± 20 cpm-1 µg protein-1 6 h). A significant increase in TßR protein in preantral follicles was evident following EGF exposure, but the effect was differential and was closely correlated with changes in mRNA levels. TßR protein in granulosa cells of preantral follicles exists in the membrane and in the cytoplasm (Fig. 3, A and B). EGF stimulated cytosolic TßRI protein expression for S3 and S4, but significant increase in the membrane form of TßRI was noted for S6 only (Fig. 3A). The cytosolic form of TßRI for S6 decreased with a corresponding increase in the membrane-associated form (Fig. 3A). EGF increased the levels of cytosolic form of TßRII for S4 and S5 and increased the levels of the membrane-associated form of TßRII for S6 (Fig. 3). Unlike for TßRI, no decrease in the cytosolic form of TßRII protein was evident (Fig. 3B).
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EGF and TGFß Interaction Influencing Follicular DNA and Progesterone Synthesis
During the 24-h culture period, EGF significantly stimulated progesterone production at most of the stages (Fig. 4). For the 48-h culture, EGF-exposed follicles maintained high levels of progesterone production during the second 24-h incubation period without EGF, but TGFß1 did not affect EGF-induced progesterone production for most of the stages (Fig. 5). TGFß1 alone had no effect on progesterone production. During the second 24-h incubation period without EGF, EGF-preexposed follicles at S1–S3 and S6 continued DNA synthesis (Fig. 6); however, TGFß1 significantly (P < 0.05) attenuated EGF-induced DNA synthesis for S1–S3 and S6 (Fig. 6).
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Effect of -Amanitin on EGF-Induced TßR mRNA in Cultured Hamster Follicles
To determine whether the increase in TßR mRNA following EGF exposure was due to TßRI and TßRII mRNA synthesis or to an inhibition of mRNA degradation, follicles from S1+2–S6 were incubated in the presence of both EGF and -amanitin. EGF exposure resulted in receptor gene transcription, as was evident from the significant reduction in receptor mRNA following a block in transcription (Fig. 7, A and B). EGF exposure for 12 h maintained the increased levels of TßR mRNA; however, administration of -amanitin (a blocker of mRNA transcription [30, 31]) resulted in a significant (P < 0.05) reduction in mRNA levels for both TßRI and TßRII (Fig. 7, A and B), suggesting that a major mechanism of EGF modulation of TßR mRNA increase is mRNA synthesis.
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DISCUSSION
The results of the present study provide the first evidence for EGF stimulation of TßR mRNA and protein expression in preantral ovarian follicles. EGF and TGFß are critical intraovarian growth factors for follicular development from preantral to antral stages [9], and TGFß has been shown to influence granulosa cell activity [1]. Hamster TßRI and TßRII have been partially cloned, and their expression has been shown to be regulated by gonadotropins [18], but little is known about the interaction between EGF and TGFß in influencing follicular development. In the present study, we demonstrated that the expression of TßR mRNA and proteins in the hamster follicles is differentially regulated by EGF depending on the stage of follicular development, suggesting that follicles at different stages may require different exposures to EGF. The results corroborate observations of EGF stimulation of TßRII protein expression in human preantral follicular cells vitro [12]. The increase in the level of TßR protein effected by EGF in S6 relative to other preantral stages suggests that EGF may prepare follicles for TGFß action, depending on their hierarchy in the developmental process. This hypothesis is supported by the fact that TGFß reduces EGF-stimulated DNA synthesis only after EGF preexposure. In the hamster, S6 follicles correspond to the largest preantral follicles from which antral follicles for ovulation are recruited during the estrous cycle [32].
The results of the present study suggest that TßRI and TßRII mRNA are expressed in preantral granulosa cells and in thecal cells of large preantral follicles. The increase in thecal TßR mRNA levels effected by EGF occurred at only the 50 ng/ml dose level, suggesting that in contrast to granulosa cells, thecal response requires an optimum level of EGF signaling. Whereas lower doses of EGF can be considered suboptimal in stimulating thecal TßR mRNA expression, the effect of the higher dose may be a combination of inhibition of TßR mRNA synthesis, stimulation of mRNA degradation, and downregulation of EGF receptor, otherwise increases in TßR mRNA levels should have occurred before downregulation of the EGF receptor. This contention is supported by the fact that EGF was added only once at the beginning of the culture, and hamster theca expresses very low levels of EGF receptor immunofluorescence (unpublished results). Because preantral follicles grow as a result of granulosa cell proliferation and thecal cells appear primarily before the formation of the antral cavity [26, 32], increased sensitivity of granulosa cells to EGF is likely. EGF mRNA and peptide and EGF receptor protein are present in granulosa cells of many species [21–25, 33], including hamster preantral and small antral follicles [1].
The physiological role of EGF in follicular development has been well documented [34–38]. Because EGF is primarily mitogenic to follicular cells [19, 39, 40], the involvement of other differentiation-inducing factors is essential for granulosa cell differentiation. In the hamster, primary preantral follicles are capable of progesterone production, and their ability to synthesize androgen and estrogen develops gradually as they progress through higher stages of development [29]. Cells of all stages of preantral follicles undergo DNA synthesis during the estrous cycle [28] and in response to exogenous FSH [41, 42]. Therefore, it is likely that follicular cells undergo many cycles of proliferation and differentiation rather than a single period of proliferation followed by a final period of differentiation. Our results suggest that EGF not only stimulates granulosa cell proliferation but may also prepare these cells to undergo subsequent TGFß-induced differentiation by modulating TßR mRNA and protein expressions.
Significant stimulation of follicular progesterone production by EGF observed in the present study confirms earlier findings [19, 39]. Similar to the present findings, EGF stimulated progesterone production by mouse granulosa cells in culture, but TGFß had no effect [43]. EGF stimulates DNA synthesis [19, 20], and TGFß attenuates EGF-induced DNA synthesis in hamster preantral follicles [10]. EGF preexposure during only the first 24 h of culture (first half) is able to maintain DNA synthesis for some stages of follicles during the second 24 h of culture (second half); however, TGFß1 attenuates EGF-stimulated DNA synthesis for every instance. Transforming growth factor ß1 by itself fails to stimulate DNA synthesis during the second half of the culture and has no effect on DNA synthesis if the stimulatory effect of EGF is absent. These results provide strong evidence that the EGF effect is a prerequisite for TGFß action on preantral follicles, at least in the hamster, in which preantral folliculogenesis occurs through precise stages of development [26]. Because steroids do not induce follicular DNA synthesis in the hamster [42], it is logical to assume that EGF-stimulated progesterone in this culture system does not affect follicular DNA synthesis.
Increased availability of receptor mRNA is a prerequisite for increased receptor protein, which determines the magnitude of TGFß action on follicular cells. In the hamster ovary, substantial amounts of TßR protein exist in the cytoplasm rather than in the membrane [8]. Therefore, increased synthesis of receptor protein may not necessarily indicate enhanced TGFß action; rather, translocation of receptor to the membrane may form an additional important regulatory mechanism. Our results indicate that EGF not only stimulates TßR mRNA for the synthesis of receptor protein but may also modulate the receptor protein levels in the membrane and cytoplasm, hence functioning as an important regulator of TGFß action on follicular cells. The decrease in EGF-stimulated TßRI and TßRII mRNA levels in almost all stages of preantral follicles following -amanitin exposure suggests that one of the mechanisms whereby EGF enhances TGFß action on follicular cells is the stimulation of TßR mRNA synthesis. The decrease in TßR mRNA levels in control cultures after -amanitin exposure suggests that endogenous stimulus differentially maintains TßRI and TßRII mRNA levels by maintaining the basal rate of mRNA transcription, by minimizing RNA degradation, or by both mechanisms. The results of the present study also suggest that a sequential interplay of EGF-TßR (EGF stimulated) and TGFß may be necessary for the growth and functional differentiation of follicles during preantral development. Further studies are in progress to determine how EGF affects TßR mRNA expression.
FOOTNOTES
First decision: 19 January 2001.
1 This study was supported by a grant (HD28165) from NICHHD and the Olson Foundation of Omaha. A portion of this work has been presented at the 33rd Annual Meeting of the Society for the Study of Reproduction at Madison, Wisconsin. 
2 Correspondence: S.K. Roy, Departments of OB/GYN and Physiology and
Biophysics, University of Nebraska Medical Center, 984515 Nebraska
Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Accepted: April 20, 2001.
Received: December 28, 2000.
REFERENCES
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