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a Department of Obstetrics and Gynecology, University of Connecticut Health Center, Farmington, Connecticut 06030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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While these possibilities are not mutually exclusive, evidence to support the concept of a hormone-growth factor cascade has been developed. Many studies have shown that gonadotropins act directly through their receptors within GCs to promote steroidogenesis [4]. In addition, gonadotropins stimulate both thecal cells and GCs to secrete various growth factors, which in turn modulate ovarian steroidogenesis [5]. Epidermal growth factor (EGF) is a growth factor that not only promotes GC progesterone (P4) secretion [6, 7] but also inhibits GC apoptosis [7]. However, the anti-apoptotic action of EGF is not observed in the presence of either the P4 receptor antagonist, RU-486 or the P4 synthesis inhibitor, aminoglutethimide [7]. Aminoglutethimide blockage of EGF's action can be overcome by P4 [7]. Collectively, these data indicate that 1) there exists a gonadotropin-induced cascade of hormones and growth factors that ultimately maintains GC viability, and 2) P4 is an important component of this anti-apoptotic cascade.
Attempts to understand the actions of P4 are complicated by the fact that ovarian follicles are composed of two different sizes of GCs [6, 8, 9]. Small GCs are relatively undifferentiated and have a limited steroidogenic capacity [6, 8, 9]. Large GCs are steroidogenic, capable of secreting both estrogen and P4 [6, 8, 9]. Interestingly, only large GCs undergo apoptosis in vitro [7]. These findings are consistent with the hypothesis that P4 regulates large GC viability via an autocrine mechanism, not requiring the presence of small GCs. The first series of studies in our present report were designed to explore the autocrine hypothesis by determining whether large GCs were able to bind P4. The binding studies revealed that only small GCs bind P4. This observation argues against an autocrine mechanism and suggests that P4 mediates its action through a paracrine mechanism. Basic fibroblast growth factor (bFGF) is important in regulating ovarian function, influencing a wide range of biological functions. These include promoting bovine GC mitosis [10, 11] and attenuating FSH-induced steroidogenesis from bovine [12], chicken [13], and rat [14] GCs. In addition, bFGF inhibits rat GC apoptosis [15, 16]. Since bFGF plays a role in regulating rat GC apoptosis and P4 induces bFGF expression in uterine cells [17, 18], our second series of studies focused on the relationship between P4 and bFGF.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female Wistar rats (22 days of age) were obtained from Charles River Laboratory (Wilmington, MA) and housed under controlled conditions of temperature, humidity, and photoperiod (12L:12D; lights-on at 0700 h). On the day of the experiment, immature animals were 23 days of age. The rats were killed by cervical dislocation between 0930 and 1000 h, the ovaries were removed, and GCs were isolated. This protocol was approved by the Animal Care Committee of the University of Connecticut Health Center.
Granulosa Cell Isolation and Culture
GCs were isolated according to the procedure of Lederer et al. [9]. For the apoptosis experiments, large GC populations were isolated by Percoll-gradient centrifugation [9]. The large GC population included approximately 20% small GCs. For all experiments, the GCs were washed, resuspended in RPMI-1640, and plated in Lab-Tek 8-chamber slides (Nunc Inc., Naperville, IL).
All steroids were purchased from Sigma Chemical Co. (St. Louis, MO), made as stock solutions in ethanol at 1 mg/ml, and then diluted in RPMI-1640 to the desired final concentration. P4 receptor (PR) antibody (C-262; StressGen, Victoria, BC) was added to cultures at a final concentration of 10 µg/ml except for the fluorescein isothiocyanate (FITC)-BSA-P4 binding study, in which the concentration was increased to 20 µg/400 µl. Depending on the experimental design, bFGF (5 ng/ml) or a goat anti-human bFGF antibody (100 µg/ml) were added to the cultures. Basic FGF and its antibody were purchased from R&D (Minneapolis, MN). Aminoglutethimide was purchased from Sigma Chemical Co. and was used at a concentration of 0.5 mM. RU-486 was provided by Rousel-UCLAF (Romainville, France) and was used at a concentration of 64 µM.
Localization of P4 Binding Sites Using FITC-BSA-P4
GCs were plated at 1.5 x 105 cells/400 µl and incubated for 30 min at 37°C in 5% CO2 in air with RPMI-1640 (ethanol-supplemented control medium) or medium supplemented with either P4 (35 µg) or PR antibody (20 µg). FITC-BSA-P4 (10 µg; Sigma Chemical Co.) was then added to the medium, and the cells were incubated for an additional 30 min. GCs were then washed twice with PBS and observed under phase and epifluorescent optics with the FITC filter set and a 40x SPlan objective (0.70 n.a.). Both phase and fluorescent images were captured with a Dage 72 CCD camera (Dage-MTI, Michigan City, IN) as grey-scale images, and the images were stored in a computer. The density units for the fluorescent images were linear over a range of 0–255 U/pixel. The relative amount of FITC-BSA-P4 binding was assessed by determining the fluorescent intensity of each small GC using IP Lab-Spectrum software (Signal Analytics Corp., Vienna, VA). The cell size (area) and mean fluorescent intensity (fluorescent intensity per pixel) of each cell was determined from the fluorescent images, with phase images used to verify the position of each cell. The background fluorescent intensity was then calculated for each treatment and subtracted from the mean fluorescent intensity of each cell. A similar study was also conducted in which FITC-BSA-P4 binding was assessed in the absence (ethanol control) or presence of 200 µM P4, estradiol-17ß, or testosterone.
A confocal microscopy study was conducted to determine whether FITC-BSA-P4 exclusively binds to the surface membrane. For this study, GCs were exposed to FITC-BSA-P4 for 30 min as previously described and then washed with fresh medium and incubated for an additional 15 min. The cells were observed under a Zeiss confocal laser scanning microscope (CLSM 410; Zeiss, Oberkochen, Germany) using an oil achrostigmat 40x (1.3 n.a.) objective with an excitation at 488 nm and an LP 515 filter. As a control, GCs were incubated with FITC-BSA-P4 and nonlabeled P4 as previously described.
Detection and Quantitation of bFGF mRNA byIn Situ Hybridization
After 24 h of culture in either control or P4-supplemented medium, GCs were fixed with 10% formalin in PBS for 5 min. Formalin was removed with two rinses of PBS. Slides were then treated with 2% Tween in deionized water for 30 min and then rinsed in running tap water for 30 min. Slides were washed in 95% ethanol twice for 5 min each, rinsed twice with deionized water, and dried overnight on a slide warmer at 42°C in a dust-free environment.
Each slide was prehybridized at 42°C for 30 min with 50 µl of 55% formamide, 6-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate), and 5-strength Denhardt's solution supplemented with 100 µg herring sperm DNA. In situ hybridization was carried out using the following 5' digoxigenin-labeled bFGF antisense oligonucleotide prepared by National Biosciences (Plymouth, MN): 5'-GAC ACA ACC CCT CTC TCT TCT GCT TC-3'. This probe was previously described [19]. As a control, a 5' digoxigenin-labeled non-sense oligonucleotide was used [20]. The slides were processed as described by Shapiro et al. [20]. Basic FGF mRNA was revealed by the presence of a blue staining reaction product. Because of the extremely low background, cells were observed and photographed with the iris diaphragm closed in order to visualize both blue-stained and nonstained cells. Once the location of all cells was determined, the iris diaphragm was opened, and images were captured as previously described [20]. These images were used to quantitate the amount of blue reaction product (i.e., bFGF mRNA). Previously, it has been shown that the amount of staining was proportional to the amount of mRNA detected (r2 = 0.9).
Immunocytochemical Localization of FGF Receptors
GCs were plated at 1 x 105 cells/well for 2 h, rinsed once with PBS, fixed with 10% buffered formalin for 10 min, and then rinsed twice in PBS. FGF receptors were localized using the Kirkegaard and Perry Histomark Streptavidin HRP kit (Kirkegaard and Perry, Gaitherburg, MD) and the Histomark Orange horseradish peroxidase conjugate reporter system. Incubations and Tris-buffered saline (pH 7.6) washes were done at room temperature. Briefly, cells were incubated with serum provided in the kit for 15 min to reduce nonspecific staining. The serum was then removed, and the GCs were incubated with an antibody to FGF Receptor (1:1000; UBI, Lake Placid, NY) for 1 h. Cells were then washed three times for 5 min per wash. Biotinylated antibody was then added, and the cells were incubated for 1 h. The cells were subsequently washed three times. Streptavidin was added, and the cells were incubated for an additional 1 h and again washed three times. Color development proceeded for 5 min using a substrate solution of diaminobenzidine (DAB). The chambers were removed from Lab-Tek slides, and the slides were dehydrated in ethanol, washed in Histoclear, and coverslipped with Permount (Fisher Scientific Co., Pittsburgh, PA). The presence of FGF receptors was detected by an orange-brown stain. As a negative control, cells were stained as outlined above with the exception that the primary antibody was omitted.
Assessment of Apoptosis
Large GC fractions were plated at a plating density of 7 x 104 cells/400 µl. GCs were cultured in RPMI-1640 (control) supplemented with P4 (64 nM), bFGF antibody, or P4 plus bFGF antibody. Cells were incubated for 24 h at 37°C in 5% CO2 in air and then stained with hydroethidine (14 µg/ml of PBS; Polyscience, Inc., Warrington, PA) [7]. The cells were then observed under epifluorescence using the rhodamine filter set, and the percentage of apoptotic single cells was determined as previously described [7]. Only single cells were evaluated, since the viability of aggregated cells is maintained through a P4-independent mechanism [21]. A similar study was conducted in which GCs were cultured with control medium supplemented with bFGF in the presence or absence of aminoglutethimide, RU-486, or PR antibody. To control for nonspecific effects of either the bFGF or PR antibody, some control and treatment groups were supplemented with the appropriate amounts of IgG. Finally, studies were conducted using BSA-conjugated P4 and dexamethasone. These steroids were used at a concentration of 64 nM.
Statistical Analysis
Except for those involving apoptosis, all experiments were run in duplicate. Apoptosis studies were conducted in quadruplicate. Each experiment was replicated at least three times. Where appropriate, data were evaluated by an ANOVA followed by a Student-Neuman-Kuels procedure. A value of p 
0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since FITC-BSA-P4 was not observed within the nuclei of small GCs, it was possible that P4 mediated its action through an interaction with a membrane-associated protein. To test this hypothesis, the ability of a cell-impermeable form of P4, BSA-conjugated P4, to prevent GC apoptosis was assessed. As shown in Figure 4, BSA-conjugated P4 was as effective in preventing GC apoptosis as P4. Further, P4's anti-apoptotic actions could not be mimicked by dexamethasone (Fig. 4).
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When the non-sense probe was used in the in situ hybridization protocol, GCs did not stain (Fig. 5A) However, the antisense bFGF probe detected mRNA for bFGF in 10 ± 3% of small GCs that were cultured under control conditions (Fig. 5, B and C). P4 increased the percentage of small GCs that expressed bFGF to 37 ± 1% (Fig. 5C; p < 0.05) and increased the amount of bFGF mRNA expressed per cell to nearly twofold (p < 0.05; Fig. 5D). To determine whether P4 mediates its action by stimulating bFGF synthesis, GCs were cultured with control medium supplemented with P4, bFGF antibody, or both. In this study, P4 suppressed apoptosis, and this action was completely attenuated by the neutralizing antibody to bFGF (Fig. 6). This antibody has also been shown to neutralize bFGF's anti-apoptotic effects [16].
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Since the possibility exists that bFGF could stimulate P4 synthesis, we assessed the ability of bFGF to maintain GC viability in the presence of aminoglutethimide, a P4 synthesis inhibitor, and RU-486, a PR antagonist. Neither of these agents inhibited the ability of bFGF to prevent GC apoptosis (Fig. 7). Similarly, the PR antibody did not influence the anti-apoptotic action of bFGF (Fig. 7).
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The previous data indicate that bFGF mediates P4's anti-apoptotic actions. Although rat GCs express FGF receptors [22, 23], studies have not determined whether both small and large GCs express these receptors. To address this issue, immunocytochemical studies were conducted. These studies revealed that FGF receptors were expressed by both small and large GCs (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Caution must be used in proposing the existence of a membrane receptor for P4. An alternative possibility could be that P4 acts though a glucocorticoid receptor, which is present within the rat ovary [32] and can be activated or antagonized by P4 and RU-486, respectively [33]. Recent studies have shown that P4 down-regulates the expression of 20
-hydrosteroid dehydrogenase within rat luteal cells and that this action is mediated through the glucocorticoid receptor [32]. While it is likely that some of P4's intraovarian actions are transduced through the glucocorticoid receptor, the present study demonstrates that dexamethasone does not mimic P4's anti-apoptotic action. Similarly, previous studies have shown that dexamethasone does not have P4's anti-mitotic activity [34]. On the basis of these studies, it is unlikely that P4's anti-mitotic and anti-apoptotic actions in rat GCs are mediated by the glucocorticoid receptor. Rather, the data to date suggests that a membrane receptor for P4 exists and is expressed exclusively by small GCs.
Since FITC-BSA-P4 binds only to small GCs, P4 appears to act directly on small GCs. This direct action could account for P4's ability to prevent the insulin-induced expression of the immediate/early genes, c-fos and c-jun [35], and small GC mitosis [34]. In contrast to its direct action on small GCs, P4 most likely maintains the viability of large GCs through a paracrine mechanism that is mediated by bFGF. This concept is supported by the following observations. First, P4 does not bind to large GCs. Second, P4 selectively acts on small GCs to increase the amount of bFGF mRNA synthesized per cell as well as the number of small GCs that express bFGF mRNA. Third, the neutralizing antibody to bFGF completely prevents P4 from maintaining the viability of GCs. In addition, the bFGF-activated signal transduction pathway does not require enhanced P4 secretion to maintain GC viability. This conclusion is based on the findings that neither aminoglutethimide, an inhibitor of P4 synthesis; RU-486, a PR antagonist; nor the PR antibody abrogate bFGF's ability to preserve large GC viability. Collectively, these observations indicate that P4 induces bFGF synthesis within small GCs. Basic FGF then binds to its receptors on the surface membranes of large GCs and activates a signal transduction pathway that preserves large GC viability.
The relationship between P4 and bFGF expression may play a central role in regulating GC viability, thereby preventing follicular atresia in vivo. As follicles develop, their ability to synthesize P4 increases [4]. This increase in P4 parallels the increase in bFGF expression [19]. On the basis of the present in vitro studies, it is likely that the increase in bFGF expression that occurs during follicle growth is due to the increase in intrafollicular levels of P4. Similarly, studies by Rider and associates [17, 18] reveal that P4 stimulates bFGF expression in the stromal cells of the uterus. These in vivo studies support the concept that the P4-bFGF paracrine relationship, observed in vitro, also exists in vivo. In vivo, P4's role as a physiological survival factor has been confirmed by the observations that RU-486 treatment induced follicular cysts and/or atresia when injected into either intact adult rats [36] or diethylstilbestrol (DES)-primed hypophysectomized immature rats [37].
The present study demonstrates that P4 promotes bFGF expression, which in turn maintains GC viability. However, the paracrine relationship between P4 and bFGF is only a small segment of a complex hormonal and growth factor cascade that regulates the viability of GCs in vivo. This cascade can be divided into steroidogenesis-enhancing and steroidogenesis-independent segments. Most of the anti-apoptotic hormones and growth factors that have been identified to date enhance steroidogenesis. For example, both FSH and LH prevent GC apoptosis and promote P4 secretion [1]. In addition, FSH also stimulates estrogen synthesis, and estrogen has been shown to prevent GC apoptosis in vivo [38]. In addition, gonadotropins stimulate the synthesis of growth factors such as epidermal growth factor (EGF). EGF could facilitate GC P4 secretion in vitro, with P4 secretion accounting for EGF's anti-apoptotic effects [7]. P4 secretion from rat GCs is also increased by other anti-apoptotic growth factors such as transforming growth factor [6], vasoactive intestinal peptide [39], growth hormone [40], and interleukin-1ß [41]. It is not known whether these growth factors mediate their anti-apoptotic action solely by increasing P4 synthesis. Conversely, insulin and insulin-like growth factor I (IGF-I) reduce P4 production but prevent GCs of cultured follicles from undergoing apoptosis [42]. Interestingly, both insulin and IGF-I increase bFGF synthesis in nonovarian tissue [43]. The possibility exists, then, that insulin and/or IGF-1 increase bFGF expression within small GCs, thereby bypassing the P4-dependent segment of the anti-apoptosis cascade. While the present study identifies the P4-bFGF axis as a central component of the paracrine communication network that maintains GC viability, it remains to be determined whether bFGF is the last growth factor in this hormonal-growth factor cascade. Further, it is important to appreciate that there may also exist additional anti-apoptotic pathways that do not involve either P4 or bFGF.
One such mechanism is mediated by N-cadherin cell contact [16, 21]. This cell contact mechanism is sufficient to maintain cell viability independent of various survival factors such as P4 or bFGF [16, 21]. However, cell contact is not always maintained between GCs of healthy follicles. For example, cell contacts are likely to be reduced during mitosis or as a result of an increase in the size of the antrum. It is proposed that at times when GC contact is reduced, exposure to various survival factors (i.e., P4, bFGF, etc.) maintains GC viability until cell contact is reestablished.
This paracrine interaction between small and large GCs, which is mediated by P4 and bFGF, is important in that it predicts that small GCs are essential for the survival of large GCs. Interestingly, both small and large GCs are present within the same follicle [8], but small GCs do not appear to die via an apoptotic mechanism [7]. The factors that regulate small GC viability have not been identified. In this regard, the nonapoptotic death of the small GCs could eventually lead to the large GCs' undergoing apoptosis. Further, selective destruction of large GCs by activation of the apoptotic pathway could result in the proliferation of small GCs, since their proliferation is negatively regulated by the P4 [34], which is secreted by the large GCs [6, 8, 9]. These two predictions are important to our overall understanding of follicular function and require intensive testing and validation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 860 679 1228; peluso{at}nso2.uchc.edu
Accepted: September 9, 1998.
Received: December 2, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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