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Ovary |
Departments of Obstetrics and Gynecology and Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-4515
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, follicle, follicular development, growth factors, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, insulin-like growth factor (IGF) I, TGFß, and growth differentiation factor 9 [1–5] may influence FSH action on granulosa cells. Cell type-specific expression of TGFß1 and TGFß2, two important members of the TGFß superfamily of ligands [6], occurs in hamster ovarian cells [7]. Ovarian expression of TGFß has also been reported for other species [2, 8–10]. Whereas TGFß2 is predominantly expressed in hamster granulosa cells and is upregulated by FSH [11], TGFß receptor type I (TßRI) and type II (TßRII) are expressed in granulosa cells, and the expression of both types is induced by FSH [7]. FSH increases TßRI and TßRII mRNA levels in hamster ovarian cells, and the effect is markedly attenuated by dexamethasone [12]. Granulosa cell functions may be influenced by juxtacrine and paracrine effects of other ovarian cell types, but whether glucocorticoid directly affects granulosa cell TßR mRNA levels and the possible mechanism(s) involved remain unknown. Transcriptional regulation of TßR gene expression may be a major regulatory mechanism modulating TGFß action on follicular cells. Both TßRI and TßRII genes have an inverted CCAAT box motif at the 5' flanking region [13, 14] that binds nuclear factor (NF) Y [15, 16], which is an evolutionarily conserved ubiquitous factor consisting of NF-YA, NF-YB, and NF-YC subunits [17–19]. Whereas dimerization of NF-YB and NF-YC is essential for NF-YA interaction, NF-YA influences transcriptional activity at multiple levels, such as increasing the DNA affinity of other transcription factors that bind to the neighboring enhancer elements, participating in the correct positioning of other transcription factors, and interacting with the TATA binding protein (TBP) complex and proteins that have intrinsic histone acetyltransferase activity [20–23]. Chang et al. [15] reported that binding of transcription factor CBFa1 (also NF-YA) to the inverted CCAAT box motif induces TßRI promoter activity in osteoblast cells, and glucocorticoid interferes with the process by suppressing NF-YA expression. Similarly, Park et al. [24] demonstrated that inhibition of histone deacetylase activity in MCF-7 or ZR-25 human breast cancer cells results in the induction of TßRII promoter activity by the recruitment of the PCAF protein, which is a transcriptional coactivator with intrinsic histone acetyltransferase activity, to the NF-YA complex that binds to the inverted CCAAT box in the TßRII promoter. However, Kelly et al. [16] documented that NF-YA binding to the consensus DNA binding site in mouse embryonal carcinoma cells reduces TßRII reporter gene expression, but binding of activating transcription factor 1 (ATF-1) to the positive cis-regulatory element of TßRII promoter results in strong expression of the reporter gene construct. Similarly, the binding of Sp3 transcription factor to TßRI or TßRII promoter in MCF-7E cells leads to significant reduction in promoter-reporter activity [25]. Collectively, transcription of the TßR gene appears to be regulated by a concerted action of positive and negative transcriptional regulators. Therefore, hormones that influence the levels of these transcription factors in granulosa cells probably will affect TßR mRNA levels and consequently TGFß action on cells. It is not clear, however, whether the levels of any of these transcription factors in preantral follicular cells are influenced by FSH, estrogen, or glucocorticoid. We investigated whether FSH or estradiol would affect follicular TßRI and TßRII mRNA levels by upregulating NF-YA or downregulating Sp3 transcriptional coactivators in preantral follicles at different stages of development, and whether dexamethasone would inhibit the effect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries were removed on the morning of proestrus (Day 4) when serum FSH levels were the lowest [26]. Preantral follicles at stages 2–4 (stages 2–4 = two to four layers of granulosa cells [GCs], respectively; stage 5 = five or six layers of GC without theca [27]) were isolated by enzymatic dissociation [27], and follicles at stage 6 (seven or eight layers of GC and theca [27]) and stage 7 (follicles with incipient antrum [27]) were microdissected. Follicles at different stages were cultured (50 at stages 2–4, 20 at stage 5, and 10 each at stage 6 and stage 7) separately in 1 ml Dulbecco modified Eagle medium supplemented with ITS+ (final concentration: 6.25 µg insulin, 6.25 µg transferrin, and 6.25 ng selenium with 5.35 µg/ml linoleic acid), 10 units of penicillin G, 10 µg streptomycin sulfate, 0.25 µg amphotericin B solution (Invitrogen, Carlsbad, CA), and 50 mg BSA at 37°C in a Haereus incubator under 5% CO2 in air for 24 h.
Experiment 1 was conducted to determine whether dexamethasone influenced FSH- or E2-induced increase in TßRI and TßRII receptor mRNA levels. Follicles were incubated without or with 0.235 IU/ml of FSH or 36 nM/ml E2. The effect of FSH or E2 was challenged with a simultaneous administration of 100 nM dexamethasone. Follicles were retrieved, rinsed in ice-cold PBS (pH 7.4), and stored at -80°C until used in RT-PCR analysis.
Experiment 2 was conducted to determine whether dexamethasone, in addition to affecting TßR transcript levels, interferes with the function of the receptor in granulosa cells. Follicles were incubated with 10 ng/ml TGFß2 in the absence or presence of 100 nM dexamethasone and 1 µCi/ml [3H]-thymidine (TdR). Following culture, follicles were used for the quantification of TdR incorporation as an index of follicular DNA synthesis.
FSH stimulates follicular DNA synthesis and TßR induction. Therefore, experiment 3 was conducted to determine whether dexamethasone influences other key functions of FSH in preantral follicles. Follicles were incubated with 0.235 IU/ml FSH, 10 ng/ml TGFß2, or 100 nM dexamethasone, either alone or combined, and 1 µCi/ml TdR. Following culture, follicles were used for the quantification of TdR incorporation in the DNA as an index of follicular DNA synthesis. Medium was saved for determination of the levels of progesterone (P) and E2 produced by follicles in response to test factors.
Because Sp3 and NF-YA/NF-YB regulate TßR gene expression in various cell lines [17], experiment 4 was conducted to evaluate whether FSH or E2 influence the levels of any of these transcriptional modulators as a potential mechanism for upregulating TßR mRNA levels and whether dexamethasone interferes with the process. For this experiment, we used both in vivo and in vitro approaches.
For the in vitro approach, follicles at stages 6 and 7 were incubated with 0.235 IU/ml FSH with or without 100 nM dexamethasone for 24 h and were then used for immunoblotting detection and quantification of Sp3 and NF-YA. For the in vivo approach, adult hamsters were hypophysectomized on Day 1 (estrus) and injected s.c. on the 10th day with twice daily doses of 5 µg FSH or 0.1 ml of 0.5% BSA/saline as a vehicle control for 2 days before ovary retrieval. Some of the FSH-treated hamsters also received a single s.c. dose of 0.5 mg dexamethasone 24 h before ovary retrieval. Another group of hypophysectomized hamsters were injected s.c. with a single dose of 0.5 mg estradiol-cipionate without or with a simultaneous s.c. injection of 0.5 mg dexamethasone at a different location or were given a single s.c. injection of 0.5 mg dexamethasone. Ovaries were collected 48 h after the first FSH injection and 24 h after the steroid injection and then snap frozen on dry ice for immunofluorescence detection of NF-YA expression in follicular and ovarian cells.
Semiquantitative RT-PCR Analysis of Follicular Tß RI, Tß RII, and S4 Ribosomal Protein mRNA Levels
Total follicular RNA was extracted using Tri-Reagent according to the procedure described by Roy [12] and Yang and Roy [28], and 0.3 µg RNA was used for semiquantitative RT-PCR analysis of receptor and S4 mRNA as described previously [12]. S4 mRNA was selected because no change in the expression patterns was noted between stages of follicles or following any treatment. Southern hybridization was performed according to the method of Schatz et al. [29] and as described previously [12]. The signal was quantified using a Packard cyclone PhosphorImager and Optiquant software (Perkin-Elmer, Norwalk, CT). After background subtraction, total digital light units (DLU) for the receptor and S4 mRNA was reduced to DLU/min, and the results were expressed as the DLU ratio of the receptor mRNA relative to S4 mRNA.
Measurement of TdR Incorporation into Follicular DNA and Follicular Production of P and E2
TdR incorporation was performed essentially as described by Roy and Greenwald [30]. The results were expressed as counts per minute of TdR per nanogram of DNA. Levels of P and E2 accumulated in the medium during in vitro culture were measured using steroid-specific RIAs as described previously [11, 31]. The intra- and interassay coefficient of variations were 5% and 10%, respectively. Steroid levels were expressed as picograms of steroid per milliliter.
Immunoblotting of Follicular Sp3 and NF-YA Protein
The general protocol of immunoblotting was essentially similar to that described previously [7, 28]. Follicles were sonicated in ice-cold 1x RIPA buffer (PBS, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 0.1 ml protease inhibitor cocktail (Sigma) and centrifuged at 26 000 x g for 15 min at 4°C to prepare the lysate. Because of the small amount of follicular material, no attempt was made to separate the nuclear fraction. Equal amounts of protein from each stage and treatment group were fractionated in 10% polyacrylamide gels [32], electrotransferred to nitrocellulose, and probed overnight with rabbit polyclonal Sp3 or NF-YA antibody in blocking buffer. Jurkat cell lysate was used as a positive control for Sp3 and NF-YA. Following chemiluminescence substrate (WestFemto; Pierce Chemical) reaction, the signal was quantified in a UVP gel documentation system and analyzed with Labworks 4.0 software (Perkin-Elmer). The membrane was then stripped of the antibody using an antibody stripping solution (Pierce), blocked overnight at 4°C, and reprobed with a monoclonal ß-tubulin antibody (Sigma). The quantitative data were normalized against ß-tubulin signal to correct for any variation in gel loading and to determine the specificity of dexamethasone effect and were expressed as optical density (OD) of respective transcription factor relative to ß-tubulin. Because NF-YB antibody barely detected NF-YB in a relatively large amount (50 µg) of stage 6 follicular protein during antibody optimization and verification, NF-YB was not quantified in experimental samples because only a limited amount of protein for each stage was available.
Immunofluorescence Detection of NF-YA Expression in Follicular Cells
Based on the preliminary results, immunofluorescence detection was performed only for NF-YA. Frozen sections 7 µm thick were fixed sequentially for 3 min each in ice-cold methanol and acetone, fixed for 10 min in freshly prepared 4% paraformaldehyde in PBS, pH 7.4, and rinsed three times in PBS at room temperature. Sections were blocked with 10% normal donkey serum (Jackson Immunoresearch, West Grove, PA) in PBS, pH 7.4, containing 0.1% Triton X-100 for 1 h at 4°C, followed by a brief rinse in ice-cold PBS and 2-min fixation in freshly made ice-cold 1% paraformaldehyde in PBS. After rinsing three times in PBS at room temperature, sections were incubated overnight with 4 µg/ml rabbit anti-NF-YA IgG or nonimmune rabbit IgG (for control) in blocking buffer at 4°C. Fluorescence signal was generated by incubating sections with 10 µg/ml donkey anti-rabbit IgG (Jackson Immunoresearch) Alexa 488 (Molecular Probes, Eugene, OR) for 1 h at 4°C. After thorough rinsing in PBS, nuclei were counterstained with 2 µg/ml propidium iodide for 5 min at room temperature, rinsed, mounted with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL), and evaluated under epifluorescence in a DMR research microscope (Leica) equipped with an Optronics Magnafire digital camera (Optronics, Goleta, CA).
For capturing the digital images, the exposure time was adjusted using sections incubated without the primary antibody to subtract any nonspecific or autofluorescence. The signal obtained after background correction was considered the antigen-specific signal. All sections for a specific receptor antigen were evaluated under identical camera settings so that comparison could be made between groups. Immunofluorescence localization studies were repeated at least three times using tissues from different animals to verify the reproducibility of the data. Representative sections were digitally photographed. Photomicrographs were arranged using Adobe Photoshop (San Jose, CA) image editing software without any further adjustment to maintain the true nature of the findings. Fluorescence signal specific for NF-YA was digitized using NIH Image 1.6 software, and the data were expressed as optical density per pixel. Because the intensity of NF-YA immunofluorescence did not vary significantly in granulosa or theca cells across follicles in different stages of development within a group, the data reflect the overall signal intensity in granulosa, theca, or interstitial cells. The mean optical density ± SEM for the interstitial cells was calculated from three different sections representing three different ovaries and for granulosa and theca cells from at least eight follicles in preantral through antral stages present in those same sections.
Statistical Analysis
All experiments were repeated at least three times using follicles from different hamsters to get a sample size of 3 and to ensure reproducibility of the data. Quantitative data were analyzed by one- or two-way ANOVA with Scheffé post hoc test using StatView 4.1 (Abacus Concepts, San Jose, CA). The level of significance was set at 5%.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3- to 4-fold; Fig. 1). Dexamethasone alone did not display a consistent influence on the basal levels of receptor mRNA, but it significantly attenuated both FSH and E2 stimulation of increase in receptor mRNA levels, especially levels of TßRII (Figs. 1 and 2).
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Because dexamethasone displayed such a strong negative influence on TßR mRNA levels, we investigated whether dexamethasone interferes with effect of TGFß on preantral follicular cells. TGFß2 significantly (P < 0.05) stimulated DNA synthesis regardless of the stage of follicular development (Fig. 3); however, dexamethasone failed to attenuate TGFß2-induced DNA synthesis. FSH significantly (P < 0.05) stimulated DNA synthesis in follicles at all stages (Fig. 4), and its effect on follicles at stages 2–4 was increased further by TGFß2 (Fig. 3). Whereas dexamethasone alone had no effect on basal DNA synthesis (same as control), it significantly attenuated FSH-stimulated increase in DNA synthesis in follicles at all stages (Fig. 4); however, the effect was blocked by coadministration of TGFß2 (Fig. 3). Because FSH also controls steroidogenic functions of preantral follicles [33], we investigated whether dexamethasone interferes with FSH-induced follicular P and estrogen synthesis in vitro. In contrast to its effect on FSH-induced DNA synthesis, dexamethasone significantly (P < 0.05) stimulated FSH-induced P synthesis at all stages (Fig. 4) and estrogen synthesis at stage 7 (Fig. 5). Although TGFß2 did not affect FSH-induced P production, it completely blocked dexamethasone stimulation of FSH action (Fig. 4) and significantly (P < 0.05) attenuated FSH-induced, FSH + dexamethasone-induced, and basal follicular E2 production (Fig. 5).
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Because dexamethasone inhibited TßR mRNA levels in preantral follicles at all stages, the levels of transcription factors that might be influenced by dexamethasone were assessed in follicles at later stages, i.e., stages 6 and 7 together. This approach was necessary to obtain an adequate amount of protein for immunoblot detection of transcription factors. Because of the small number of GCs in preantral follicles, it was not feasible to prepare nuclear extract. The Sp3 antibody detected a 100-kDa and a 60-kDa band in all samples (Fig. 6). However, no change in Sp3 expression was noted following FSH exposure with or without dexamethasone (Fig. 6). According to the manufacturer, the antibody to NF-YA should detect 35- and 40-kDa bands in positive controls. In our study, the NF-YA antibody detected a 40-kDa protein in Jurkat cell lysate and a 35-kDa protein in NRK cell lysate (Fig. 7A). The NF-YA antibody detected both protein bands in hamster preantral follicular lysate, confirming the specificity of the antibody. The basal expression of 40-kDa NF-YA was low and was not influenced by dexamethasone treatment (Fig. 7, A and B). FSH significantly (P < 0.05) increased the levels of 40-kDa NF-YA protein, but the effect was suppressed by dexamethasone (Fig. 8, A and B). Similarly, the expression of the 35-kDa NF-YA protein increased significantly (P < 0.05) following FSH exposure, but it was markedly attenuated by dexamethasone (Fig. 7, A and B).
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For immunofluorescence photomicrography, antigen-specific signal was overlaid on nucleus-specific signal to identify nuclear or nonnuclear localization of the NF-YA. Because NF-YA-specific fluorescence was green and the corresponding nuclear signal was red, overlays were yellow. The shade of the yellow became more greenish with increasing expression of NF-YA protein and finally became green when very high levels of transcription factor were present.
Immunofluorescence studies revealed that NF-YA is expressed exclusively in the nuclei of granulosa and interstitial cells and in oocytes of primordial (S0) and primary (S1) follicles (Fig. 8, A–H). NF-YA immunoreactivity decreased noticeably in the oocyte when follicles reached the secondary stages (i.e., S2–S6; Figs. 8B and 9A). Distinct NF-YA signal was present in GCs of primordial follicles (arrowheads, Fig. 8A). Basal levels of NF-YA remained in GCs following hypophysectomy, but only a few interstitial cells displayed NF-YA signal and the intensity was modest (Figs. 8A and 9B). FSH administration for 48 h resulted in a marked increase in NF-YA immunoreactivity in GCs; however, theca cells wherever present and interstitial cells adjacent to follicles also displayed intense immunofluorescence signal (Figs. 8B and 9B). Marked increase in NF-YA signal was also noted in mural and antral GCs and in cumulus cells of large antral follicles (S10); however, NF-YA signal in the oocyte was modest (Fig. 8C). Most notably, E2 stimulated NF-YA expression in granulosa as well as in interstitial cells beyond that observed in FSH-treated ovaries (Figs. 8D and 9B). No NF-YA-specific immunofluorescence was detected when E2-treated ovary sections were incubated without the antibody, indicating the specificity of the antibody and immunofluorescence signal (Fig. 8E). Differential staining of granulosa versus interstitial cells in hypophysectomized hamster ovaries also confirmed the immunolocalization data.
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Dexamethasone alone did not significantly affect basal NF-YA expression (Figs. 8F and 9B); however, it markedly reduced both FSH- and E2-induced increase in NF-YA immunosignal in granulosa, theca, and interstitial cells (Figs. 8, G and H, and 9B). The decline was more pronounced in the interstitial cells of E2-treated ovaries (Fig. 8H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the results of this study cannot definitively determine whether NF-YA actually stimulates TßR promoter activity in GCs, based on the evidence obtained in other cell types this action is highly likely. Nevertheless, our data do not support the hypothesis that Sp3 is a transcriptional repressor of TßR in GCs. Because analysis of promoter activity requires cell culture, the present model of intact preantral follicles is not compatible with that approach. Results of the present study provide strong evidence that the effect of glucocorticoid on TßR mRNA levels in preantral granulosa cells is not mediated by other nonfollicular cells in the ovary. The basal steady-state levels of receptor mRNA, even after 24 h of culture without any hormonal supplements, indicate that TßR mRNA is stable and any increase following FSH or E2 administration reflects the transcription of the receptor gene. The different basal levels of TßRI and TßRII mRNA in preantral follicles across stages indicate that receptor subtype expression varies as preantral follicles grow under endogenous hormonal conditions. The different expression patterns of TßR mRNA following FSH or E2 administration clearly demonstrate that preantral follicles at all stages are not identical in functions and regulation. The stronger negative impact of dexamethasone on TßRII mRNA levels suggests that FSH (at least partly via E2) or E2 regulation of TßRII gene expression may be more critically controlled by glucocorticoid. This contention stems from the fact that TßRII binds to TGFß ligands [37]; hence, regulation of the levels of this subtype may form the first step for precise control of TGFß action on follicular cells, particularly as preantral follicles develop. The presence of intense NF-YA-specific signal in primordial and primary oocytes despite the absence of TßR mRNA or protein [7] (unpublished results) suggests that NF-YA may be involved in the expression of genes important for early oocyte development. Conversely, NF-YA may act as a repressor for certain genes whose expression is vital for oocyte development and maturation in later stages of follicle development. Specific studies are needed to address this issue.
FSH-mediated GC proliferation involves EGF and TGFß in the hamster [4, 38, 39], rat [40, 41], and pig [42, 43] and IGF-I in the pig [44]; therefore, glucocorticoid regulation may reflect an important mechanism that governs this intricate process. Whereas TßR mRNA levels decrease following the gonadotropin surge [12], TGFß2 protein expression in preantral GCs increases [11], coinciding with increased DNA synthesis [45]. These lines of evidence and the results of the present study suggest that although dexamethasone reduces TßR mRNA levels, it does not alter existing TßR protein in GCs, at least during the initial phase of the culture, thus allowing TGFß to bind its receptor and counteract the effects of dexamethasone on FSH-stimulated DNA and steroid hormone synthesis. This lack of effect is further evident from the inability of dexamethasone to influence the FSH effect in the presence of TGFß2. Therefore, glucocorticoids probably affect TGFß action on preantral follicles by modulating TGFß receptor gene expression rather than postreceptor signaling mechanisms of TGFß. However, dexamethasone enhancement of FSH-stimulated follicular steroid production and attenuation of FSH- and estrogen-mediated NF-YA synthesis indicate that glucocorticoids modulate FSH and estrogen receptor activation and/or downstream signaling necessary for these biological responses. Recently, Imasato et al. [46] and Shuto et al. [47] demonstrated that dexamethasone upregulates Toll-like receptor 2 expression in HeLa cells by suppressing p38-mitogen-activated protein kinase (MAPK) via MAPK phosphatase 1 upregulation. Although our data document a dexamethasone-induced suppression of FSH/estrogen-stimulated TßR gene expression in GCs, the involvement of one or more glucocorticoid-modulated intermediary factors cannot be ruled out. The expression pattern of NF-YA appears to be one such factor; however, additional studies are needed to address this issue and the mechanism thereof in detail.
The lack of TGFß2 effect on FSH-induced follicular P production but its attenuation of FSH-induced E2 production suggests that TGFß differentially influences FSH-regulated follicular steroidogenesis. TGFß augments FSH-stimulated P production by rat GCs in vitro [48], but it inhibits FSH-stimulated P production by porcine GCs [42]. Such discrepancy in TGFß action may be due to species variation. The synergism of dexamethasone and FSH in follicular P production and its inhibition by TGFß2 indicates that glucocorticoid and TGFß interaction may be necessary for GC differentiation as the follicle matures through the preantral stages. Hsueh and Erickson [49] demonstrated that either cortisol or dexamethasone significantly stimulates P production by rat GCs in culture, thus corroborating our findings. Although glucocorticoid inhibits FSH-induced increase in aromatase activity, it does not affect the activity of preexisting aromatase activity in rat GCs [49]. A similar situation may explain the lack of dexamethasone effect on follicular E2 synthesis in the hamster.
These results provide strong evidence to suggest that inhibition of NF-YA expression is one of the mechanisms whereby glucocorticoids suppress TßRI and TßRII mRNA levels in hamster preantral follicles. A model explaining the effect of FSH, estrogen, TGFß, and dexamethasone interaction with reference to TßR and NF-YA expression during follicular development has been proposed (Fig. 10). Future studies will focus on the intracellular signaling pathway that is interrupted by dexamethasone to bring about reduced NF-YA expression.
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FOOTNOTES |
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2 Correspondence: Shyamal K. Roy, Departments of Obstetrics and Gynecology and Physiology and Biophysics, BH 4030, University of Nebraska Medical Center, 984515 Nebraska Medical Center, Omaha, NE 68198-4515. FAX: 402 559 6164; skroy{at}unmc.edu
Received: 1 November 2002.
First decision: 20 November 2002.
Accepted: 15 January 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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