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BOR - Papers in Press, published online ahead of print March 8, 2006.
Biol Reprod 2006, 10.1095/biolreprod.105.050294
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BIOLOGY OF REPRODUCTION 75, 140–148 (2006)
DOI: 10.1095/biolreprod.105.050294
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Transforming Growth Factor B1 Stimulated DNA Synthesis in the Granulosa Cells of Preantral Follicles: Negative Interaction with Epidermal Growth Factor1

Peixin Yang 3 4, and Shyamal K. Roy 2 45

Departments of OB/GYN4 and Cellular and Integrative Physiology,5 University of Nebraska Medical Center, Omaha, Nebraska 68198-4515

ABSTRACT

EGF or TGFB1 alone stimulates but together attenuate granulosa cell DNA synthesis. Intact preantral follicles from hamsters were cultured with TGFB1, EGF, or both to reveal the mechanisms of such unique regulation. Follicular CCND2 (also known as cyclin D2), CDKN1B (also known as p27kip1), and the involvement of appropriate signaling intermediaries and kinases were examined. TGFB1, acting via SMAD2 and SMAD3, antagonized the degradation of CCND2 protein by blocking its phosphorylation. In contrast, TGFB1 supported CDKN1B degradation by involving MAPK14 (also known as p38 Map Kinase) and PKC, resulting in CDK4 activation and DNA synthesis. EGF via MAPK3/1 maintained functional levels of CCND2 through CCND2 synthesis as well as degradation. EGF and TGFB1 together inhibited CDK4 activation and DNA synthesis. EGF attenuated TGFB1 stimulated phosphorylation of SMAD3, TGFB1-induced activation of MAPK14 and PKC, and TGFB1 suppression of CCND2 degradation. In contrast, TGFB1 suppressed EGF-induced increase in CCND2 mRNA levels. The final outcome was CCND2 degradation without replenishment and decreased activities of MAPK14 and PKC leading to suppression of CDK4 activation. The results indicate that each growth factor involves a separate mechanism to maintain an effective level of CCND2 in granulosa cells for the activation of CDK4 and induction of DNA synthesis. However, their simultaneous action is inhibitory to follicular DNA synthesis because they counteract each other's activity by interfering at specific sites. Because both EGF and TGFB1 are present in granulosa cells, this mechanism may explain how their effects are temporally modulated for granulosa cell proliferation and folliculogenesis.

DNA synthesis, EGF, follicle, granulosa cells, growth factors, ovary, signal transduction, TGFB1

INTRODUCTION

TGFB1 has an antiproliferative effect on epithelial cells [1], but it stimulates DNA synthesis in mesenchymal cells [2]. TGFB1 stimulates DNA synthesis in rat granulosa cells in culture [3] and hamster granulosa cells within intact preantral follicles [4]. TGFB1 also stimulates the growth of mouse preantral follicles [5]. Although TGFB1 alone has little or no effect on the DNA synthesis in porcine [6, 7] or bovine [8] granulosa cells, it suppresses EGF-induced DNA synthesis in porcine [6] and bovine [8] granulosa cells in culture. However, May and Schomberg [7] have shown that TGFB1 at a 1-ng/ml dose level potentiates EGF or EGF plus IGF1-induced DNA synthesis in porcine granulosa cells in culture and EGF-induced increase in cell number in the presence of low concentration of platelet-poor serum [7]. These lines of evidence suggest that despite a species-specific variation in the effect of these growth factors on the granulosa cells, EGF and TGFB1 interaction may be important for proper follicular development. TGFB1 and its cognate receptors [9] are expressed in hamster granulosa cells, and the expression is regulated by FSH and steroid hormones [10]. Signaling via TGFB1 receptors includes phosphorylation of SMAD2 and SMAD3 [11], which are present in granulosa cells [12–15]. TGFB1 action on many nonovarian cell types leads to activation of MAPK3/1, MAPK14, or PKC pathways [11]. Whether any or all of these signaling mechanisms are involved in TGFB1-induced DNA synthesis in granulosa cells remains unclear. FSH stimulates both EGF [16] and TGFB1 [4] expression in the granulosa cells of preantral follicles. The expression of EGF and TGFB1 and EGFR in the granulosa cells increases following the preovulatory FSH surge [17, 18], which stimulates DNA synthesis in preantral follicles [19]. In contrast, TGFBR levels markedly decline during the surge [9], which is expected to render TGFB1 momentarily ineffective. Because both EGF and TGFB1 are expressed in follicular cells, it is important to understand whether their combined effect is necessary for granulosa cell DNA synthesis or the growth factors that sequentially modulate each other function to regulate this process.

CCND2 couples with CDK4 to initiate DNA synthesis in granulosa cells [20, 21], and the process can be inhibited by CDKN1B, which is an endogenous CDK inhibitor [22]. TGFB1 inhibits epithelial cell proliferation by upregulating CDKN2B (also known as p15INK), CDKN1A (also known as p21Cip1), and CDKN1B [1]. We have reported that in contrast to synchronized, serum-dependent cell cultures, granulosa cells within intact preantral follicles do not require CCND2 synthesis to initiate DNA synthesis [21]; rather, CCND2 synthesis occurs as a follow-up to offset CCND2 degradation [21]. Further, activation of MAPK3/1 is essential for CDK4 activation in granulosa cells [21]. Whether TGFB1-stimulated DNA synthesis in granulosa cells involves similar mechanisms is not known. Taken together, these lines of evidence suggest that the mechanisms underlying EGF and TGFB1 interaction in regulating granulosa cell DNA synthesis need to be determined in order to understand how they impact preantral follicular development in the ovary. Therefore, the objectives of the present study were to reveal the mechanisms of TGFB1-induced DNA synthesis in the granulosa cells of intact preantral follicles and to reveal the combinatorial effect of EGF and TGFB1 on the process.

MATERIALS AND METHODS

Golden hamsters (90–100 g; Sasco, Madison, WI, and Charles River laboratories, Charles River, MA) were kept in a 14L:10D cycle in a climate-controlled facility according to the Institutional Animal Care and Use Committee (IACUC) and USDA guidelines. The use of hamsters for this study was approved by the IACUC.

Polyclonal rabbit antibodies to CCND2, CDK4, MAPK3/1, and UB (also known as ubiquitin, all forms) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal rabbit antibodies to CDKN1B and SMAD2/3 and recombinant murine epidermal growth factor (EGF) were from BD/Transduction Laboratories (Lexington, KY); polyclonal rabbit antibodies to phospho-SMAD2, phospho-PKCA/PKCB1 (also known as PKC {alpha}/ßII), MAPK14, phosphoMAPK14 (Thr180/Tyr182) and phospho-threonine, and monoclonal mouse dual-phosphoMAPK3/1 were from Cell Signaling Technology (Beverly, MA); rabbit polyclonal anti-phospho-SMAD3 was a generous gift from Dr. Edward Leof (Mayo Clinic, Rochester, MN); monoclonal anti-mouse TUBB (also known asbetatubulin) was from Sigma Chemical Co. (St. Louis, MO); [3H] thymidine (specific activity 40 Ci/mmol) was from Amersham (Arlington Heights, IL); recombinant human TGFB1 was from R&D Systems (Minneapolis, MN); purified retinoblastoma (RB1) protein, myelin basic protein (MBP), and enzyme assay kits for MAPK14 and PKC (all isozymes) were from Upstate Biotechnology, Inc. (Upstate, NY); oligodeoxynucleotide primers were synthesized by Eppley Molecular Biology Core facility at UNMC; [32P]-{gamma}-ATP (specific activity 7000 Ci/mmol) was from ICN Radiochemicals (Costa Mesa, CA); and PD98059, SB 203580, and bisindolylmaleimide I (GFXI) were from Calbiochem (La Jolla, CA).

The study was done using intact preantral follicles at stage 6 (seven to eight layers of granulosa cells and a thin layer of theca) and stage 7 [23] from proestrous hamsters. Granulosa cells without any thecal contamination were used whenever indicated.

Experiment 1: Effect of TGFB1 on Follicular DNA Synthesis and CCND2 Degradation

Follicles were cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) with 1% ITS+ (final concentration per ml: 6.25 µg insulin, 6.25 µg transferrin, 6.25 ng selenium, 5.35 µg linoleic acid, BD Biosciences), 1% antibiotic mixture (penicillin, streptomycin, and Amphotericin B; Invitrogen), and 0.5% bovine serum albumin at 37°C under 5% CO2 in air [24] with 1 µCi/ml [3H]thymidine and increasing concentrations of human TGFB1 for indicated times. Control culture received no treatment. Follicles were sonicated in a kinase assay buffer as described previously [21], and the incorporation of [3H]thymidine; levels of CCND2, CDKN1B, and TUBB; and the activity of the CDK4 were determined [21, 25]. The steady-state levels of CCND2 mRNA relative to ACTB (also known as beta-actin) mRNA were determined by a semiquantitative RT-PCR using follicular RNA as previously reported [21]. Negative controls for RT-PCR were reverse transcription without RNA or with RNA but without MMLV reverse transcriptase. Positive control was ovarian RNA from proestrous hamsters. Because [3H]thymidine incorporation reached a steady-state level by 6 h with 5 ng/ml TGFB1 (see results), all subsequent studies were done in the presence of 5 ng/ml TGFB1.

To determine whether follicular [3H]thymidine incorporation included thecal contribution, follicles were cultured with or without 5 ng/ml TGFB1 and [3H]thymidine for 6 h as described earlier. Thecal covering was ruptured, and granulosa cells were squeezed out in ice-cold PBS. Thecal shell was everted, and any residual granulosa cells were removed as best as possible and rinsed three times with ice-cold PBS, and both cell types were assayed for [3H]thymidine incorporation and the results expressed in terms of DNA.

To determine TGFB1 effect on cyclin degradation, follicles were cultured for 3 h with 5 ng/ml TGFB1 and sonicated in a UB (also known as ubiquitin) protein detection buffer (see below). CCND2 was immunoprecipitated from 100 µg of total protein using 4 µg of CCND2 antibody and sequentially immunoblotted with 1:1000 dilution of the UB and CCND2 antibodies. All samples were clarified using an equal amount of nonimmune IgG of appropriate host species and protean A agarose as described previously [21]. The rationale for 3 h of exposure was to detect CCND2 protein before it was degraded to nondetectable levels.

Experiment 2: Signaling Events in TGFB1-Induced DNA Synthesis

Follicles were cultured for 1 h with 10 µM PD98059 [21], 100 nM GFXI [26], or 100 nM SB203580 [27] to block MAPK3/1, PKC, or MAPK14 activity, respectively, before the administration of 5 ng/ml TGFB1 and [3H]thymidine. Follicles were retrieved 6 h later, and the incorporation of [3H]thymidine and CDK4 activity was determined. Next, follicles were cultured for 6 h in the presence of TGFB1 or EGF, and MAPK3/1 phosphorylation was examined by immunoblotting.

Next, to determine the time course of TGFB1-induced phosphorylation of SMAD2, SMAD3, MAPK14, and PKCA/B1 in granulosa cells, follicles were cultured with or without TGFB1 for indicated times, granulosa cells isolated as indicated previously, and levels of phosphoproteins determined by sequential immunoblotting and densitometry. The rationale for using granulosa cells was to exclude any thecal contribution to SMAD immunoblots because TGFB1, although it did not stimulate thecal DNA synthesis, could affect other thecal functions [28].

Experiment 3: Effect of EGF Plus TGFB1 on Follicular DNA Synthesis

To determine the combined effect of EGF and TGFB1 on follicular DNA synthesis, follicles were cultured for 6 h with or without EGF, TGFB1 (or both), and [3H]thymidine. Levels of CCND2 mRNA, CCND2 protein and CDKN1B protein, CDK4 activity, and [3H]thymidine incorporation were determined.

Next, follicles were cultured for 3 h with or without EGF, TGFB1, or both to determine a possible mechanism of CCND2 protein degradation. CCND2 was immunoprecipitated from follicular homogenate and the amount of UB associated with CCND2 antibodies determined by immunoblotting with 1:1000 dilution of anti-UB antibody. Next, threonine-phosphorylated proteins were immunoprecipitated from follicular homogenate, and levels of phosphorylated CCND2 were detected by immunoblotting with 1:1000 dilution of the antibody.

Based on the time course of SMAD2 and SMAD3 phosphorylation, follicles were cultured for 1 h in the presence or absence of EGF, TGFB1, or both; granulosa cells were separated from the theca; and phosphoSMAD2, phosphoSMAD3, SMAD2/3, phosphoMAPK3/1, MAPK3/1, phospho-MYC, MYC, and TUBB were detected by sequential immunoblotting. The dilution of phosphoMAPK3/1 antibody was 1:2000, but all other antibodies were diluted to 1000-fold. We observed that EGF-induced MAPK3/1 phosphorylation in the granulosa cells occurred after 10 min of EGF exposure in vitro and remained stable for 6 h (unpublished observation). Phosphorylation of MYC as an early response gene product was correlated with EGF-induced DNA synthesis [29, 30]. Therefore, Myc phosphorylation was examined to reveal whether TGFB1-induced granulosa cell DNA synthesis would mimic EGF-like activity.

The mechanisms of negative interaction between EGF and TGFB1 were further examined by culturing follicles for 3 h in the presence or absence of either EGF, TGFB1, or both; granulosa cells were separated from the theca, and the activities of MAPK14 and PKC in granulosa cell lysate were measured according to the protocols supplied by the manufacturer. The enzyme activity was calculated as picomol [32P]-phosphate transferred to the substrate per minute per mg protein, and the data were expressed as fold changes relative to untreated controls. The 3-h time period was selected on the basis of the time course of enzyme activation by TGFB1.

Determination of [3H]Thymidine Incorporation, CCND2 mRNA, and Immunoblotting

[3H]Thymidine incorporation, immunoprecipitation, immunoblotting, and kinase assays were done as described previously [19, 21]. Immunoblot signal was developed with ECL Advanced kit (Amersham Biosciences, Piscataway, NJ) and captured directly using a UVP gel documentation system (Upland, CA). The intensity (optical density) of the band was quantified and expressed in terms of TUBB or immunoprecipitated protein. The homogenization buffer and all kinase assay buffers contained 200 µM sodium orthovanadate and a protease inhibitor cocktail. The homogenization buffer for UB protein detection contained 10 µM N-ethylmaleimide and 50 µM N-acetyl-leucyl-norleucinal [21] to block proteosomal activity.

Steady-state levels of CCND2 and ACTB mRNA were determined by RT-PCR amplification as described previously [21].

Statistical Analysis

All cultures were repeated at least three times. Each culture had 50 follicles per group, and there were three replicates per group. Representative immunoblots of three separate analyses were presented. The optical density of the immunoblots relative to TUBB or immunoprecipitated protein were presented whenever appropriate. Each bar represented a mean and a standard error (SEM) of three detections. All quantitative data were analyzed by one-way ANOVA with the Scheffé post hoc test using the StatView (Abacus Concepts, Inc, Berkeley, CA) statistical software program. Comparison was done between the control and experimental groups for each subset of determination. The level of significance was 5%.

RESULTS

Effect of TGFB1 on Follicular DNA Synthesis

The goal was to determine whether TGFB1-induced follicular DNA synthesis would require upregulation of CCND2 protein. Both 1- and 5-ng/ml doses of TGFB1 activated CDK4 and stimulated [3H]thymidine incorporation by 6 h (Fig. 1A). A dose of 0.5 ng/ml could also increase [3H]thymidine incorporation but required 12 h of exposure, whereas doses below 0.5 ng/ml had no effect, and doses beyond 5 ng/ml did not produce any further increase in [3H]thymidine incorporation (data not shown). Whether theca cells contributed to follicular thymidine incorporation was examined by measuring thymidine incorporation in both cell types. Basal [3H]thymidine incorporation was low for the theca, but, in contrast to granulosa cells, the incorporation did not increase in response to TGFB1 (Fig. 1B), suggesting that follicular DNA synthesis-related changes in signaling proteins would reflect primarily granulosa cell activities. Further, the results also indicated a differential effect of TGFB1 on follicular cell types. Therefore, intact follicles were used in subsequent experiments to avoid unnecessary delays in sample processing. Consistent with DNA synthesis, increased levels of CCND2 and decreased levels of CDKN1B protein (Fig. 1C) were evident, but CCND2 mRNA levels remained unaltered (Fig. 1D). UB immunoblotting revealed that compared to untreated controls, virtually no UB protein was associated with CCND2 protein when follicles were exposed to TGFB1, and consequently, more nondegraded CCND2 protein was immunoprecipitated (Fig. 1E).


Figure 1
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  		FIG. 1. Effect of TGFB1 on follicular DNA synthesis. A) Time course analysis of CDK4 activity and [3H]thymidine incorporation in preantral follicles cultured with different doses of TGFB1. RB1 was used as a substrate for CDK4. B) Incorporation of [3H]thymidine in the granulosa and thecal cells in response to 5 ng/ml TGFB1. Intact follicles were cultured for 6 h, and granulosa cells were separated from the theca. C) Immunoblot analysis of CCND2 and CDKN1B in follicles cultured with 5 ng/ml of TGFB1 for various times. The bottom panel represents the optical density (OD) of the blots. D) Semiquantitative RT-PCR analysis of CCND2 and ACTB mRNA expression in follicles cultured with 5 ng/ml of TGFB1 for various times. E) Effect of TGFB1 on UB binding to CCND2 in follicles cultured with 5 ng/ml of TGFB1 for 3 h. The top panel represents the levels of UB associated with CCND2, the middle panel represents the corresponding amount of CCND2, and the bottom panel represents the OD. Each bar is a mean of at least three values ± SEM. P < 0.05: bars with a different letter; P > 0.05: bars with the same letter.

Involvement of PKCA/B1 and MAPK14 in TGFB1-Induced DNA Synthesis

Because FSH-stimulated DNA synthesis in hamster follicles involved MAPK3/1 activation [21], whether TGFB1 would activate MAPK3/1 in follicular cells was also examined. Inhibition of MAP2K1 by PD98059 had no effect on TGFB1-induced DNA synthesis and CDK4 activation (Fig. 2A), and in contrast to EGF, TGFB1 did not phosphorylate MAPK3/1 (Fig. 2B). However, TGFB1-induced CDK4 activation and DNA synthesis were significantly attenuated by GFXI and SB203580, either separately or combined (Fig. 2A). SB203580 appeared to have a stronger effect (Fig. 2A). Interestingly, GFXI prevented TGFB1-induced decrease in CDKN1B but did not affect CCND2 protein levels (Fig. 2C). Identical results were obtained when SB203580 was used instead (data not shown). The optimal dose of each inhibitor was selected on the basis of a dose-response analysis using preantral follicles. In a cell-free system, each inhibitor at the selected dose level specifically blocked the activity of the target enzyme. Next, the time course of SMAD2, SMAD3, PKCA/PKCB1, and MAPK14 phosphorylation was examined to reveal the latency of TGFB1 activation of these signaling intermediaries in the granulosa cells of preantral follicles. In a preliminary study, we found that PKCA and PKCB1 were the only isozymes that were phosphorylated by TGFB1 in hamster follicles (unpublished observation); hence, we selected these isozymes. Although TGFB1 phosphorylated both SMAD2 and SMAD3 after 30 min, only SMAD2 remained phosphorylated during a 3-h culture (Fig. 3, A and B). SMAD3 phosphorylation peaked at 1 h and then declined to the baseline level at 2 h (Fig. 3). Although the levels of phosphorylated MAPK14 started to increase after 30 min, significant increase was evident only after 1 h. The levels of phosphorylated enzyme decreased slightly but significantly after 2 h (Fig. 3B). On the contrary, the levels of phosphorylated PKCA and PKCB1 showed an upward tendency after 2 h of TGFB1 administration (Fig. 3B), but significant increase was evident after 3 h (Fig. 3, A and B).


Figure 2
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  		FIG. 2. Effect of PKC, MAP2K1, or MAPK14 inhibitor on TGFB1-induced follicular DNA synthesis. Follicles were cultured for 6 h with 5 ng/ml TGFB1 with or without the enzyme inhibitor. A) Top panel represents RB1 phosphorylation as an index of CDK4 activity, and the bottom panel represents DNA synthesis. B) Immunoblot analysis of MAPK3/1 phosphorylation in follicles cultured for 3 h with EGF or TGFB1. EGF was used as a positive control. C) Immunoblot detection of CCND2 and CDKN1B levels in follicles cultured for 6 h with TGFB1 in the absence or presence of 100 nM GFXI. The bottom panel represents the OD of protein expression. Each bar is a mean of at least three values ± SEM. P < 0.05: bars with a different letter; P > 0.05: bars with the same letter.


Figure 3
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  		FIG. 3. Time course of TGFB1 phosphorylation of SMAD2, SMAD3, MAPK14, PRKCA, and PRKCB1 in the granulosa cells of preantral follicles. A) Intact follicles were cultured with 5 ng/ml TGFB1, and granulosa cells were separated from the theca and used for immunoblotting. B) Optical densities of the immunoblots presented in A. Each bar is a mean of at least three values ± SEM. P < 0.05: bars with a different letter; P > 0.05: bars with the same letter.

Combined Effect of EGF and TGFB1 on Follicular DNA Synthesis, CCND2 Protein Degradation, and TGFB1 Signaling in Follicular Cells

The rationale was to examine whether a combination of EGF and TGFB1 would have an additive effect on follicular DNA synthesis. The rationale stemmed from the fact that these growth factors were present in follicles in vivo; hence, an interaction would be expected. Whereas EGF or TGFB1 activated CDK4 and markedly stimulated follicular DNA synthesis, their combined action completely suppressed DNA synthesis (Fig. 4A). Consistent with these findings, TGFB1 completely suppressed EGF-induced increase in follicular CCND2 mRNA levels (Fig. 4B). Interestingly, the combined action of the growth factors resulted in an almost complete elimination of CCND2 but significant upregulation of CDKN1B protein compared to the effect of EGF or TGFB1 alone (Fig. 4C).


Figure 4
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  		FIG. 4. Effects of a combination of EGF and TGFB1 on follicular (A) CDK4 activity (top panel) and DNA synthesis (bottom panel). B) Levels of CCND2 mRNA (top panel: gel image; bottom panel: OD). C) Immunoblots of CCND2 and CDKN1B proteins (top panel) and the OD of the immunoblots (bottom panel). Follicles were cultured with 50 ng/ml EGF or 5 ng/ml TGFB1 for 6 h. Each bar is a mean of at least three values ± SEM. P < 0.05: bars with a different letter; P > 0.05: bars with the same letter.

In the absence of any exogenous growth factor, a basal amount of UB protein was associated with CCND2 protein (Fig. 5A). The relative amount of UB associated with CCND2 increased significantly (P < 0.05) in follicles exposed to EGF without any change in the levels of CCND2 (Fig. 5A). In contrast, negligible amount of UB but a higher amount of CCND2 was present in follicles exposed to TGFB1 alone (Fig. 5A). The levels of CCND2 fell to the basal level with a corresponding increase in UB in follicles exposed simultaneously to both of the growth factors (Fig. 5A).


Figure 5
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  		FIG. 5. Effects of a combination of EGF and TGFB1 on CCND2 ubiquitination and phosphorylation of signaling proteins. A) Immunoblot detection of ubiquitin associated with CCND2 protein. Follicles were cultured with 50 ng/ml EGF or 5 ng/ml TGFB1 for 3 h, CCND2 was immunoprecipitated, and the levels of UB and CCND2 were detected by immunoblotting (top panel). The bottom panel represents the OD of the immunoblots. B) Immunoblot detection of threonine-phosphorylated CCND2. Threonine-phosphorylated proteins were immunoprecipitated from follicular homogenate, and CCND2 was detected by immunoblotting. Mouse IgG was used as a negative control for the phosphothreonine (phosThr) antibody. C) Phosphorylation of signaling intermediaries. Follicles were cultured for 1 h with or without 50 ng/ml EGF or 5 ng/ml TGFB1, and granulosa cells were separated from the theca and used for immunoblot detection of the phosphorylated proteins (top panel). The bottom panel represents the OD of the immunoblots. Each bar is a mean of at least three values ± SEM. P < 0.05: Bars with a different letter; P > 0.05: bars with the same letter.

Examination of CCND2 phosphorylation revealed that without any stimulus, CCND2 was modestly phosphorylated on threonine (Fig. 5B). CCND2 phosphorylation virtually disappeared in follicles exposed to TGFB1 (Fig. 5B). EGF markedly induced CCND2 phosphorylation, but it also reversed the inhibitory effect of TGFB1 (Fig. 5B), suggesting that higher UB binding to CCND2 could be because of increased phosphorylation on threonine residue. No significant amount of phosphorylated CCND2 could be immunoprecipitated from the homogenate of EGF-exposed follicles when the primary antibody was replaced with the nonimmune IgG (Fig. 5B).

We examined some key steps in growth factor signaling to determine whether the negative interaction between EGF and TGFB1 also occurred at the level of initial signal transduction. TGFB1 phosphorylated SMAD2 and SMAD3, whereas EGF phosphorylated MAPK3/1 and MYC (Fig. 5C). A small increase in MYC level was also evident following EGF exposure (Fig. 5C). Whereas TGFB1 did not interfere with EGF-activation of MAPK3/1 or MYC, EGF selectively attenuated TGFB1-induced SMAD3 phosphorylation (Fig. 5C). Further, EGF or TGFB1 significantly stimulated the activities of MAPK14 and PKC (all isozymes) in the granulosa cells (Fig. 6). However, enzyme activities declined markedly after the follicles were cultured with a combination of EGF and TGFB1 (Fig. 6).


Figure 6
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  		FIG. 6. Activities of MAPK14 and PKC in the granulosa cells of preantral follicles cultured for 3 h with EGF, TGFB1 or both. The unit of enzyme activity were referred to picomol phosphate transferred to the substrate per minute per mg protein, and the data were expressed as fold changes relative to untreated control for comparison. Each bar is a mean of at least three values ± SEM. P < 0.05: bars with a different letter; P > 0.05: bars with the same letter.

DISCUSSION

The results of the present study unveil some potential mechanisms whereby EGF and TGFB1 can modulate each other's activities to regulate granulosa cell DNA synthesis that is the key to preantral follicle growth. It is also apparent that these growth factors act via different mechanisms to stimulate DNA synthesis. A negative interaction between EGF and TGFB1 in granulosa cell DNA synthesis has been reported in the hamster, rat, pig, and cow [6–8, 31, 32], but the mechanisms remain elusive. The effect of TGFB1 on porcine granulosa cells seems to depend on the culture condition and dosages. Mondschein et al. [6] have shown that TGFB1, regardless of the dose level, suppresses EGF-induced thymidine incorporation in porcine granulosa cells collected from immature pigs and cultured in the presence of platelet extraction supplements. In contrast, May et al. [7], using granulosa cells of adult pigs, have shown that TGFB1, even at a 10-ng/ml dose, potentiates EGF-induced DNA synthesis. Further, TGFB1 enhances EGF-stimulated cell division in the presence of low levels of platelet-poor plasma-derived serum; however, it suppresses the EGF effect when the serum concentration increases. In the present study, TGFB1 alone at a 5-ng/ml dose (with the maximum dose of 20 ng/ml; data not shown) stimulates granulosa cell DNA synthesis but demonstrates negative interaction with EGF (even when present at a 0.5-ng/ml low dose level; data not shown). EGF has no effect on follicular DNA synthesis in the hamster when the EGF dose is below 50 ng/ml (unpublished observation). In porcine granulosa cell culture models, 10% fetal bovine serum is used for attachment. Besides species, other important differences between the present study and the findings on porcine granulosa cells are 1) culture of intact preantral follicles vs. granulosa cell monolayer, 2) complete absence of serum or extracellular matrix vs. the presence of serum or matrix at the beginning and/or during the study, 3) 6 h of culture vs. several days of culture, and 4) use of follicles immediately after their harvest vs. use of cells after they have seeded for days. Nevertheless, the negative interaction between EGF and TGFB1 seems to occur across species. Although the effects of two doses of TGFB1 on granulosa cell DNA synthesis have been presented, a dose-response study has indicated that even 0.5 ng/ml TGFB1 is capable of stimulating DNA synthesis, albeit with longer exposure (data not shown). However, doses lower than 0.5 ng/ml have no effect. TGFB1 levels in gonadotropin-treated human follicles range from 3 to 6 ng/ml [33] and 5 to 8 ng/ml in hamster follicular fluid (unpublished observation). Granulosa cells from porcine small antral follicles can produce 0.6 ng TGFB1/106 cells per day by the third day of culture [34]. These lines of evidence seem to suggest that intraovarian TGFB1 levels exist in the nanogram range. No attempt is made to quantify TGFB1 production by hamster preantral follicles because of technical limitations; however, based on the information, the dose used in the present study does not exceed the physiological limit. What role does the endogenous TGFB1 play in this context? The hourly production rate of TGFB1 by hamster preantral follicles is not known; however, 106 porcine granulosa cells, seeded for 3 days in the presence of serum, can produce approximately 200 pg TGFB1 during a 24-h culture [34]. Expression of TGFB1 in hamster follicles increases after the endogenous FSH surge or exogenous FSH administration [35]. Although a basal production of TGFB1 by preantral follicles cannot be ruled out, it is unlikely that the amount produced during a 6-h culture will have a significant impact on cell functions.

Previously, we have demonstrated that FSH-induced activation of CDK4 in the granulosa cells of preantral follicles does not require an immediate synthesis of CCND2; however, CCND2 mRNA levels increase afterward for a possible steady-state maintenance of the protein [21]. Increase in CCND2 mRNA levels following EGF exposure in the present study supports the contention. One of the novel findings of the present study is that whereas the degradation of CDKN1B that is important for the activation of CCNE (also known as cyclin E) [36] is a necessary step for EGF or TGFB1 to initiate DNA synthesis, EGF maintains the steady-state levels of CCND2 by stimulating its synthesis, while TGFB1 increases the levels by preventing CCND2 degradation. Degradation of both CCND2 and CDKN1B appears to be mediated by ubiquitination as suggested for other G1-phase cyclins [37, 38]. In the UB-ligase complex, the F-box protein provides substrate specificity to the ligase complex [39], thus making the UB-mediated degradation protein specific [40]. Selective degradation of CDKN1B and protection of CCND2 by TGFB1 but degradation of both proteins by EGF, suggest that these growth factors may differentially regulate protein ubiquitination. The model of intact preantral follicles has provided the opportunity to study granulosa cell response to growth factors without disrupting the cell-cell communication that is vital to maintain granulosa cell phenotype. The lack of thecal DNA synthesis in response to TGFB1 suggests that molecular changes observed herein reflect primarily granulosa cell activities, and results obtained with granulosa cells seem to support this conjecture. Similar results have been obtained when follicles were cultured with EGF (unpublished observation).

All CCND (D-type cyclins) undergo constant degradation [41]. It has been shown for CCND1 that despite overexpression of CCND1, CCND1 protein levels remain modest because of ubiquitination and rapid turnover of the protein by the PSM (also known as proteosome 26S subunit; no specific class) [42]. The modest basal levels of CCND2 protein in unstimulated follicles may reflect cellular activities that have been induced in vivo. Phosphorylation of CCND1 on threonine-286 has been shown to be necessary for the degradation of CDK4-bound CCND1 [43]; however, free CCND1 appears to be ubiquinated independently of phosphorylation on threonine-286 [39]. Because CCND1 is the predominant G1-phase cyclin in most cell lines, information about CCND2 is meager. Nevertheless, increased phosphorylation of CCND2 on threonine by EGF and its suppression by TGFB1 is consistent with the mechanism of recognition and degradation of the cyclin by the PSM. Whether the phosphorylation is on threonine-286 is not known because no antibody to threonine-286 phosphorylated CCND2 is available at present. TGFB1 inhibits proteosomal activity in a variety of cell lines [44]. The increase in CCND2 in response to TGFB1 without any increase in CCND2 mRNA suggests that the protection from proteosomal degradation may be the primary mechanism whereby TGFB1 increases CCND2 levels in granulosa cells. In contrast, EGF stimulates the synthesis as well as degradation to maintain a functional level of CCND2 in cells. Therefore, it can be speculated that CCND2 declines to a nonfunctional level when TGFB1 blocks EGF-induced CCND2 synthesis but not the degradation, resulting in a failure in CDK4 activation and DNA synthesis. Future studies may address this issue further.

FSH or EGF activates MAPK3/1 in granulosa cells to initiate DNA synthesis [21, 45, 46]. It is evident that TGFB1-stimulated DNA synthesis involves MAPK14, PKCA, and PKCB1, which seem to regulate CDKN1B degradation. Whether MAPK14, PKCA, and PKCB1 have additional roles in CDK4 activation in the context of TGFB1 action or the mechanisms whereby the enzymes affect CDKN1B degradation are not yet known. EGF seems to establish a PKC-mediated MAPK3/1 self-activation loop that is critical for CDK4 activation in hamster granulosa cells; however, the degradation of CDKN1B is an essential early step (unpublished observation). PKC activity has been shown to be necessary for TGFB-stimulated proliferation of MCF-7 cells [47] and insulin-dependent rat granulosa cells [48] and suppression of gonadotropin-induced granulosa cell differentiation [49]. Activation of MAPK14 in Jurkat cells results in increased phosphorylation of RB1 and concurrent transcriptional activity of E2F [50].

It is notable that EGF-induced impairment of SMAD3 phosphorylation is adequate to disrupt TGFB1 stimulation of granulosa cell DNA synthesis. It can be speculated that SMAD3 signaling may be important for CDKN1B degradation, protection of CCND2 from ubiquitination, and activation of PKCA and PKCB1 and MAPK14, while SMAD2 signaling may interfere with CCND2 transcription. Consistent with this conjecture, recent studies have shown that follicular development leading to ovulation is compromised in mice with intact SMAD2 but deleted SMAD3 genes [51]. The mechanisms whereby TGFB1-induced SMAD3 phosphorylation in granulosa cells is selectively diminished by EGF is the subject of a future study, but the possible involvement of specific phosphatases cannot be ruled out. The unique dichotomy in TGFB1 signaling in granulosa cells underscores the importance of EGF and TGFB1 interactions during follicular development. The expression of EGF and TGFB1 and EGF receptor in granulosa cells increases following the preovulatory FSH surge [17, 18], which stimulates DNA synthesis in preantral follicles [19]. In contrast, TGFB1 receptor levels markedly decline during the surge [9], which is expected to render TGFB1 momentarily ineffective. Receptor expression recovers by the estrous morning under the influence of the second preovulatory FSH surge [9]. Therefore, a possible lack of TGFB1 action following the surge will allow EGF to stimulate granulosa cell DNA synthesis. Subsequently, when TGFB1 becomes effective with the availability of its receptors, it attenuates the action of EGF on granulosa cell proliferation. This mechanism will allow cells to enter the postmitotic rest period and prepare for the next round of cell division and prevent aberrant cell proliferation. Intrabursal injection of TGFB1 blocks follicular growth and ovulation in mice [52]. It is possible that TGFB1 can stimulate granulosa cell proliferation later in the estrous cycle; however, its activity is likely to be modulated by EGF. In this way, follicles can grow in an orderly fashion during each estrous or menstrual cycle.

In summary, the study reveals a novel mechanism whereby TGFB1 stimulates DNA synthesis in granulosa cells of hamster preantral follicles. Further, the study identifies the differences in the mechanisms of EGF- and TGFB1-mediated granulosa cell DNA synthesis and reveals unique mechanisms whereby EGF and TGFB1 counteract each other's activity to regulate follicular DNA synthesis. Future studies may provide additional details to substantiate the present results.

ACKNOWLEDGMENTS

We thank Dr. Edward Leof of Mayo Clinic, Rochester, MN, for generously providing pSmad3 antibody, without which the study could not be completed.

FOOTNOTES

1 Supported by a grant (HD 28165) to S.K.R. P.Y. was a Lalor Foundation fellow. Back

2 Correspondence: Shyamal K. Roy, DRC 5013, Departments of OB/GYN and Cellular and Integrative Physiology, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu Back

3 Current address: Department of Internal Medicine, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205. Back

Received: 15 December 2005.

First decision: 4 January 2006.

Accepted: 7 March 2006.

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