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Biology of Reproduction 64, 163-170 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Dexamethasone Inhibits Luteinizing Hormone-Induced Synthesis of Steroidogenic Acute Regulatory Protein in Cultured Rat Preovulatory Follicles1

Tsurng-Juhn Huanga, and P. Shirley Li2,a

a Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, Republic of China

ABSTRACT

The effect of dexamethasone on LH-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of cyclic adult rats on the morning of proestrus with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 10 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of the follicles with increasing concentrations (1–1000 ng/ml) of dexamethasone suppressed LH (10 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc was not affected by this dexamethasone treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of P450scc. Dexamethasone also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10-5 M) or a cAMP analogue 8-Br-cAMP (0.5 mM). The effects of dexamethasone on 8-Br-cAMP-induced StAR protein levels and progesterone production were blocked by cotreatment of the follicles with glucocorticoid receptor antagonist RU-486. These results demonstrate that dexamethasone inhibits the LH-induced StAR protein levels and that the effects of dexamethasone are mediated by the glucocorticoid receptor.

cortisol, follicle, LH, ovary

INTRODUCTION

Glucocorticoids are hormonal mediators of stress. Previous studies on the effects of stress on reproductive performance in many species indicate that glucocorticoids adversely affect fertility [1]. Pathological conditions of the adrenal cortex such as Cushing's syndrome are associated with ovarian dysfunction, including chronic anovulation and polycystic ovarian syndrome. Elevated levels of cortisol have been reported in trained female athletes as well as in patients with anorexia and bulimia nervosa, amenorrhea being a common clinical feature of these groups [2–4]. The effects on ovarian function may occur at the hypothalamus (to decrease the synthesis and release of GnRH), the anterior pituitary gland (to inhibit the synthesis and release of gonadotropins), or the ovary (to modulate steroidogenesis and/or oogenesis). It is thought that the principal deleterious action of the glucocorticoids occurs at the hypothalamus and the anterior pituitary gland, creating a state of hypogonadotropic hypogonadism. This view is supported by a huge body of data, including observations that administration of synthetic glucocorticoids can significantly decrease hypothalamic GnRH release [5–7] and can inhibit the GnRH-stimulated release of LH and FSH from the pituitary [7–11]. However, glucocorticoid receptors have been identified in granulosa cells [12], and glucocorticoids have been shown to exert direct effects on ovarian steroidogenesis, both in vivo and in vitro [13–15]. Another mechanism by which the hypothalamic-pituitary-adrenal axis may influence reproductive function is by a direct effect of glucocorticoids on the target tissues of sex steroid production [16].

The biosynthesis of gonadal steroid hormones—progesterone, estrogens, and androgens—begins from cholesterol. The acute regulated and rate-limiting step for the synthesis of steroid hormones is the delivery of cholesterol from cellular stores to the mitochondrial inner membrane where the cholesterol is then converted to pregnenolone by cytochrome P450 side-chain cleavage (P450scc) enzyme [17, 18]. The transport of cholesterol in steroidogenic cells is thought to be mediated by steroidogenic acute regulatory (StAR) protein [19]. The StAR protein has been identified as a mitochondrial phosphoprotein [20] that is rapidly induced by tropic hormones [21, 22]. The decisive demonstration came from an inherited disease that leads to a dramatic deficiency in all steroid hormones, congenital lipoid adrenal hyperplasia: mutations in the StAR gene have been shown to underlie this disorder [23–25].

Results from recent studies have demonstrated the pattern of expression and regulation of StAR in ovarian cells during various physiological processes. High levels of StAR protein and mRNA were observed in functional corpus luteum (CL), whereas they were absent in regressed CL [26–28]. Their expression was subject to luteotropic hormones such as eCG, hCG [29], LH [30], and estradiol [31] as well as the luteolytic agent prostaglandin F2{alpha} (PGF2{alpha}) [27, 29, 30]. The StAR protein is also regulated in a gonadotropin-dependent and stage-specific manner during follicular development [26, 29, 32, 33]. Studies in vitro showed that gonadotropins and activators of the protein kinase A pathway increase StAR expression in granulosa cells [27, 32–36], whereas PGF2{alpha} and phorbol 12-myristate 13-acetate appeared to be negative regulators of StAR expression in vivo and in vitro [29, 30, 32, 33].

In view of the importance of StAR in basal and hormonally regulated steroidogenesis and the limited knowledge of glucocorticoid regulation of this gene in ovarian follicles, we have used the rat preovulatory follicles as an experimental model to investigate the effects of the glucocorticoid dexamethasone on the expression of StAR protein and progesterone production.

MATERIALS AND METHODS

Hormones and Reagents

Purified porcine pituitary LH (pLH; USDA-pLH-B-1; 1.7 U/mg) was obtained from the National Hormone and Pituitary Distribution Program (Bethesda, MD). Dexamethasone (dexamethasone phosphate) was obtained from Narn Guang Chemical Co. (Tainan, Taiwan). RU-486 (Mifepristone) was provided by Roussel-Uclaf (Paris, France). Ham F12-Dulbecco modified Eagle medium (F12-DMEM; 1:1) and other culture supplies were purchased from Gibco-BRL (Grand Island, NY). Antiserum directed against amino acids 88–98 of mouse StAR was kindly provided by Dr. D.M. Stocco (Department of Cell Biology & Biochemistry, Texas Tech University, Lubbock, TX). Rabbit anti-P450scc antiserum (provided by Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica, Nankang, Taiwan) was generated by immunization with a human P450scc fusion protein overexpressed in Escherichia coli [37].

Isolation and Dissection of Rat Preovulatory Follicles

Female Wistar rats (National Cheng Kung University Laboratory Animal Center, Tainan, Taiwan), aged 10–12 wk, were maintained in 22–24°C rooms on a 12L:12D schedule (lights-on at 0600 h). Food and water were provided ad libitum. Estrous cycles were monitored by daily vaginal smears. Animals were used after three consecutive 4-day estrous cycles. After light ether anesthesia and decapitation at 0900–1100 h on proestrus, ovaries were removed, trimmed of adhering tissues, and placed in 100-mm glass petri dishes containing F12-DMEM with 0.1% BSA (F12-DMEM-BSA) for follicle dissection. At all times, the animals were treated in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animals. Using a dissecting microscope and watchmaker's forceps, the largest preovulatory follicles (>400 µm in diameter) were dissected from the ovary and cleaned of adhering stromal tissue and/or smaller follicles.

Follicle Culture

Follicles from six to nine animals were pooled for each experiment. Each set of experiments was performed two to four times. Eight follicles were incubated per dish (35 x 10 mm), with at least three dishes used for each treatment. Follicles were randomly chosen from the pool and cultured for 24 h at 37°C in a humidified atmosphere with 5% CO2 and 95% O2 as described [38] in 1.5 ml F12-DMEM-BSA supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate with or without LH in the absence or presence of increasing concentrations of dexamethasone or as indicated. At the end of incubation, follicles were solubilized in TSE buffer (10 mM Tris, 0.25 M sucrose, and 0.1 mM EDTA, pH 7.4) for extracting mitochondrial protein for subsequent Western blot analysis, and the media were collected for the determination of progesterone level by RIA.

Western Blot Analysis

For Western blotting, follicles were homogenized in 1 ml of TSE buffer using a motor-driven glass homogenizer with a serrated Teflon pestle. Homogenates were centrifuged at 600 x g for 30 min, and the supernatant was collected and centrifuged at 12 000 x g for 30 min. Protein concentration was determined by Lowry's method [39]. The proteins (50 µg) were mixed with SDS-PAGE sample buffer (58.3 mM Tris-HCl, pH 6.8, 1.7% SDS, 1% 2-mercaptoethanol, 5% glycerol, and 0.002% bromophenol blue), boiled for 5 min, and loaded into a 12.5% mini gel by standard SDS-PAGE procedures, along with prestained molecular weight markers (Bio-Rad, Richmond, CA). Gels were electrophoresed at 50 mA/min at room temperature using a running buffer (pH 8.3) that included 25 mM Tris-base, 192 mM glycine, and 0.1% SDS. The proteins were electrophoretically transblotted to a polyvinylidene difluoride membrane (Bio-Rad) using a semidry electrotransfer apparatus (Hoefer Scientific Instruments, San Francisco, CA). Protein transfer was conducted for 40 min at 0.8 mA/cm2 in transfer buffer (pH 8.3) consisting of 20 mM Tris-base, 150 mM glycine, 10% methanol, and 0.01% SDS. The membrane was blocked by a 60-min incubation in blocking buffer (PBS buffer containing 0.5% Tween 20 and 4% nonfat dry milk), followed by a 60-min incubation with anti-StAR (1:1000) or anti-P450scc (1:2500). After three washes for 5 min each with PBS-Tween (PBST) buffer (as above, without milk), the membranes were incubated for 40 min with horseradish peroxidase-conjugated donkey anti-rabbit IgG (1:10 000) (Amersham Life Science Ltd., Buckinghamshire, England). After five washes with PBST, membranes were developed using the enhanced chemiluminescence detection system according to the manufacturer's instructions (New England Nuclear, Boston, MA). The intensity of the bands on Western blots was measured by the Arcus II computer-assisted image system (PDI Inc., Huntington Station, NY).

Progesterone RIA

Quantitation of progesterone directly from aliquots of the medium was performed by RIA as previously described [40]. The antiserum to progesterone-11{alpha}-BSA (C467-B4) was supplied by Dr. J.E. Hixon (Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, Urbana, IL) and used at the dilution of 1:25 000 in 0.1 M Tris buffer (pH 7.4). The sensitivity of the assay was 12.5 pg per assay tube.

Statistical Analysis

All values were expressed as the mean ± SEM of pooled data from two to four experiments. Two means were compared using Student's t-test. Where there were more than two means, significant differences between means were determined by ANOVA. The means were then analyzed by Fisher's probable least-squares difference multiple comparison.

RESULTS

Dose-Dependence Effect of LH on StAR Protein Expression and Progesterone Production

To determine the optimal dose of LH, preovulatory follicles were cultured for 24 h with various doses of LH, and StAR protein levels were determined. As shown in Figure 1a, a dose-dependent relationship between StAR protein expression and LH treatment was readily seen in rat preovulatory follicles as determined by Western blot analysis. The StAR protein was low in unstimulated follicles but could be seen with levels of LH as low as 0.01 ng/ml. The level of StAR protein continued to increase, reaching a maximal value at 10 ng/ml. This dose was used in the subsequent experiments.



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  		FIG. 1. Effect of increasing concentrations of LH on StAR protein levels (a) and progesterone production (b). Preovulatory follicles (>400 µm in diameter) isolated from ovaries of proestrous rats were cultured (8 per culture/1.5 ml) in serum-free medium for 24 h in the absence (C, control) or presence of LH (0.01–100 ng/ml). At the end of culture, mitochondrial protein extracts were dose-dependently prepared from 24 follicles of three cultures at each dose of LH as described in Materials and Methods. For each sample, 50 µg of mitochondrial proteins were analyzed for StAR protein by Western blotting analysis also as described in Materials and Methods. The position of the StAR protein is indicated. The StAR protein was detected by chemiluminescence and quantitated using the Arcus II computer-assisted image system at each treatment. Integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined by RIA in duplicate samples from three culture dishes at each dose of LH. Values are the mean ± SEM from four independent experiments. P < 0.05 between control and concentrations above 0.1 ng/ml.

Progesterone production in the culture medium from the same experiment is shown in Figure 1b. Luteinizing hormone at concentrations of 0.1, 1, 10, and 100 ng/ml caused a dose-dependent increase in progesterone production, reaching a maximal effect at a concentration of 10 ng/ml.

Effect of Treatment with Dexamethasone on the Expression of StAR and P450scc Proteins and Progesterone Production

To investigate the effects of dexamethasone on the expression of StAR and P450scc proteins, preovulatory follicles were incubated for 24 h with or without increasing concentrations of dexamethasone in the presence or absence of LH. As shown in Figure 2a, dexamethasone had a significant (P < 0.05) effect in suppressing LH-stimulated StAR protein expression. The inhibitory effect was concentration dependent, and a 24-h exposure of preovulatory follicles to dexamethasone concentrations ranging from 10 to 1000 ng/ml decreased StAR protein levels by 37–71%. Basal StAR protein levels were low after 24 h of culture. Treatment with dexamethasone had no effect on P450scc protein levels (Fig. 2a).



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  		FIG. 2. Effect of the dose of dexamethasone on LH-stimulated StAR and P450scc protein levels (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) LH (10 ng/ml) in the presence or absence (B; C, control) of increasing concentrations of dexamethasone (1–1000 ng/ml). The immunoblots were analyzed for StAR and P450scc proteins. The positions of the StAR and P450scc proteins are indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. Values are the mean ± SEM from two (for StAR) or three (for P450scc) independent experiments. The progesterone concentration was determined in duplicate samples from three culture dishes at each dose of dexamethasone. Values are the mean ± SEM from three independent experiments. P < 0.05 between control and concentration above 1 ng/ml

Results in Figure 2b show that dexamethasone at a concentration of 10 ng/ml or higher significantly (P < 0.05) decreased LH-stimulated progesterone production. The inhibited level was about 59–74% of that treated with LH alone (75.7 ± 18.7 ng for 8 follicles over 24 h). The basal accumulation of progesterone was 3.75 ± 1.21 ng/8 follicles per 24 h, which was 6.9 ± 3% of that in cultures treated with LH alone.

Effect of Treatment with Dexamethasone on Forskolin- and 8-Br-cAMP-Stimulated StAR Protein Expression and Progesterone Production

The influence of dexamethasone upon the ability of forskolin (10-5 M), an activator of adenylate cyclase, and 8-Br-cAMP (0.5 mM), a cAMP analog, to stimulate StAR protein expression was also investigated. Dexamethasone at a concentration higher than 1 ng/ml inhibited (P < 0.05) the stimulatory effect of forskolin on StAR protein to about 40% (Fig. 3a). Progesterone production in control cultures averaged 2.6 ± 0.4 ng for 8 follicles over 24 h and was increased (P < 0.05) by forskolin stimulation (142.0 ± 17.1 ng for 8 follicles over 24 h) (Fig. 3b). Dexamethasone at a concentration higher than 1 ng/ml reduced the stimulatory effect of forskolin (P < 0.05).



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  		FIG. 3. Effect of the dose of dexamethasone on forskolin-stimulated StAR protein levels (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) forskolin (10-5 M) in the presence or absence (B; C, control) of increasing concentrations of dexamethasone (1–1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined in duplicate samples from three culture dishes at each dose of dexamethasone. Values are the mean ± SEM from two independent experiments. P < 0.05 between control and concentration above 1 ng/ml

Results in Figure 4a show that the stimulatory effect of 8-Br-cAMP on StAR protein expression was inhibited by concomitant treatment with dexamethasone (10, 100, or 1000 ng/ml) in a dose-dependent manner, decreasing from 55.8 ± 7.7% to 18.8 ± 5.2%. Treatment with 8-Br-cAMP significantly (P < 0.05) increased progesterone production (148.6 ± 20.4 ng for 8 follicles over 24 h) as compared to basal levels (5.6 ± 1.6 ng for 8 follicles over 24 h) (Fig. 4b), whereas concomitant treatment with increasing concentrations of dexamethasone reduced progesterone production in a dose-dependent manner with 1000 ng/ml of dexamethasone decreasing progesterone production to 35%.



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  		FIG. 4. Effect of the dose of dexamethasone on 8-Br-cAMP-stimulated StAR protein levels (a) and progesterone production (b). The conditions for this experiment are identical to those described for Figure 1 with the exception that the follicles were cultured with or without (B, basal) 8-Br-cAMP (0.5 mM) in the presence or absence (B; C, control) of increasing concentrations of dexamethasone (1–1000 ng/ml). The immunoblots were analyzed for StAR protein. The position of the StAR protein is indicated. After quantitation, integrated optical density values were expressed as a percentage of that measured in mitochondria from control follicles. The progesterone concentration was determined in duplicate samples from three culture dishes at each dose of dexamethasone. Values are the mean ± SEM from two independent experiments. P < 0.05 between control and concentration above 1 ng/ml

Effect of RU-486 on Dexamethasone Inhibition of StAR Protein Expression and Progesterone Production

The glucocorticoid receptor antagonist, RU-486 [41], was used to determine whether the effects described above were mediated by the glucocorticoid receptor. Follicles were cultured for 24 h with 8-Br-cAMP (0.5 mM) in the absence or presence of dexamethasone (100 ng/ml) and with or without RU-486 (50 µM). The results are presented in Figure 5. RU-486 antagonized the inhibitory effect of dexamethasone on StAR protein levels and progesterone production.



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  		FIG. 5. Evaluation of the effects of dexamethasone (DEX) and RU-486 (RU) on 8-Br-cAMP-stimulated StAR protein levels (a) and progesterone production (b). Follicle culture, incubations, mitochondrial protein extraction, and analysis were conducted as described in Figure 1. The position of the StAR protein is indicated. Integrated optical density values were expressed as a percentage of that measured in mitochondria from 8-Br-cAMP-treated follicles. The progesterone concentration was determined in duplicate samples from three culture dishes at each treatment. Values are the mean ± SEM from two independent experiments. B, Basal. Bars with different letters differ significantly (P < 0.05)

DISCUSSION

Using a serum-free culture system of preovulatory follicles isolated from ovaries of cyclic adult rats, we have demonstrated that the glucocorticoid dexamethasone is able to decrease significantly LH-, forskolin-, or 8-Br-cAMP-induced StAR protein levels. The dexamethasone effect is dose dependent and is associated with decreases in progesterone production. These results indicate that dexamethasone inhibits preovulatory follicle steroidogenesis by down-regulating StAR protein expression. We also demonstrated that RU-486 that can displace dexamethasone from the glucocorticoid receptor reversed the effect of dexamethasone on StAR protein levels and progesterone production. This strongly suggests a glucocorticoid receptor-mediated mechanism of inhibition of 8-Br-cAMP-induced StAR protein expression. The RU-486 effect is specific, as this compound by itself does not influence the expression of StAR and progesterone production induced by 8-Br-cAMP nor does it have any effect on the basal level of StAR.

The studies reported here provide evidence for a local effect of glucocorticoids on the ovary by the direct demonstration of the inhibitory activity of dexamethasone on the production of progesterone in response to LH. To date, the direct effects of glucocorticoids on ovarian function have been investigated in isolated granulosa cells. Our present observations on isolated preovulatory follicles confirm the previous findings observed in human granulosa-lutein cells, which showed that dexamethasone inhibited LH-stimulated pregnenolone production [15]. In contrast, dexamethasone has been shown to enhance the FSH-induced production of progesterone in rat preantral primary granulosa cells [42]. The discrepancy in results may be explained by the differences in experimental conditions used, such as the status of differentiation of the follicles. Granulosa cells from immature diethylstilbestrol-treated rats might be considered nontransformed and undifferentiated or in the process of differentiation, while granulosa cells from preovulatory follicles of the adult cycling rat would be more mature and highly differentiated. It has been reported that in rat and human granulosa cells that have not luteinized in vitro, glucocorticoids can potentiate the cAMP and steroid responses to LH and FSH [13, 14, 42–44]. Thus, it would appear that the effects of glucocorticoids on the steroidogenic activity of a given ovarian cell type may enhance FSH action in granulosa cells in the follicular phase of the ovarian cycle but predominantly attenuate LH-stimulated steroidogenesis during the luteal phase. Although strict comparative studies using granulosa and thecal cells from preovulatory follicles were not performed in this study, the use of our experimental model, however, suggests that glucocorticoid in vivo may act directly on the ovary to modulate follicular steroidogenesis.

In our studies, the effective inhibitory dose of dexamethasone was at 10 ng/ml (1.94 x 10-8 M), which is less than circulating levels of cortisol found in athletic women with amenorrhea (230 ± 10 nM) [4] and women with bulimia nervosa (120 ± 40 nM) [2]. Because greater than 90% of circulating glucocorticoids are bound by transcortin [45, 46], the dose (10 ng/ml) of dexamethasone, a more potent synthetic glucocorticoid, may represent an overestimate of the amount of free glucocorticoid circulating in the blood of these disturbed women, and the effect seen is probably more pharmacological than physiological. Thus, the influence of glucocorticoids in normal follicular steroidogenesis may be minimal. However, excessive production of glucocorticoids resulting from perturbations of the hypothalamic-pituitary-adrenal axis or the administration of large amounts of glucocorticoids for therapeutic reasons has been shown to alter the normal activity of ovarian follicular development. Interruption of folliculogenesis under these conditions may be explained, at least in part, by a direct glucocorticoid action on ovarian cells. Although it has been reported that glucocorticoids can decrease hypothalamic GnRH output [5–7] and can inhibit pituitary responsiveness to GnRH and thus prevent the release of LH and FSH [7–11], the present findings challenge the dogma that adrenal hyperactivity suppresses reproductive function solely by the central withdrawal of LH and/or FSH support. Moreover, a number of studies that report a glucocorticoid-induced suppression of ovarian function in vivo without significant changes in the plasma gonadotropins [47, 48] suggest that these direct effects may supersede the central actions of glucocorticoids in disrupting ovarian function.

The inhibitory action by dexamethasone was prevented in the whole follicle by RU-486. Specific receptors to glucocorticoids are found in rat ovaries [12]. However, RU-486 also blocks progesterone receptors [41]. Studies in human granulosa cells indicate that progesterone regulates granulosa cell proliferation and differentiation in an autocrine-paracrine manner [49]. Another study of porcine granulosa cells with the synthetic progesterone R-5020 showed that the involvement of progesterone in its own production was minimal [50]. Therefore, our results indicate that the effects of dexamethasone on progesterone production in rat preovulatory follicles are mediated through the glucocorticoid receptor. Because little information is available on the synthesis of the glucocorticoid receptor and the process of downregulation of its hormone receptor in the ovary in states of glucocorticoid excess, we do not know if the regulation of the glucocorticoid receptor by dexamethasone exists. Additional studies are necessary to define further the role that glucocorticoids play in the regulation of the glucocorticoid receptor gene expression in the ovary.

Whether the inhibitory mechanism of glucocorticoids is mediated prior or distal to cAMP formation was examined by investigation of the effect of dexamethasone on forskolin- and 8-Br-cAMP-stimulated progesterone production. Dexamethasone treatment suppressed forskolin-stimulated progesterone production. This finding suggests that dexamethasone may suppress adenylate cyclase activity, resulting in the reduction of cAMP formation. In addition, to reduce cAMP formation, our observation that dexamethasone treatment also attenuated 8-Br-cAMP-stimulated progesterone production indicated that the action of dexamethasone is exerted, at least in part, at a point distal to the generation of cAMP. Further studies involving direct measurement of follicular cAMP production and the activities of adenylate cyclase are needed to elucidate this point.

With regard to the direct cellular mechanisms responsible for the effect of glucocorticoids on hormone-induced steroidogenesis, our present study demonstrates that in rat preovulatory follicles, dexamethasone acting via the glucocorticoid receptor, inhibits LH-induced progesterone production, whereas the amount of P450scc is not affected by dexamethasone treatment, indicating that the loss of steroidogenic capacity is not a result of inhibition of P450scc. Furthermore, dexamethasone decreased LH-induced, forskolin-induced, or 8-Br-cAMP-induced StAR protein levels. This suggests that in rat preovulatory follicles, the major inhibitory effect of dexamethasone is at the step of StAR. Diminished levels of StAR protein would likely result in a reduction in the capacity of preovulatory follicles for progesterone production, in response to gonadotropins, by limiting the availability of cholesterol to the P450scc complex, located in the inner mitochondrial membrane. Previous studies of the StAR protein have indicated that it has an indispensable role in steroid hormone biosynthesis [23], and it has been further postulated that this role is in regulating cholesterol transfer to the inner mitochondrial membrane [19, 20]. It appears that the dexamethasone-induced depression in progesterone production in preovulatory follicles is to be due to the inhibition of StAR protein synthesis. This observation is highly consistent with previous studies in which inhibition of steroid hormone biosynthesis has been tightly correlated with StAR synthesis. For example, agents and conditions that have been shown to result in a decrease in steroid hormone biosynthesis, such as cycloheximide [21], lipopolysaccharide [51], diethylumbelliferyl phosphate [52], PGF2{alpha} [29], and estrogen withdrawal [31], have all been demonstrated to decrease StAR protein content. Moreover, a StAR-dependent reduction in steroidogenesis could be manifested through a reduction in the expression and/or activity of the protein. It is possible that the inhibitory effect of dexamethasone on StAR protein occurs as a result of a reduction in cAMP-mediated transcription of the StAR gene and/or StAR mRNA stability. It is also possible that dexamethasone may exert a negative influence on StAR activity in preovulatory follicles through post-translational modification (e.g., phosphorylation) of the protein [53]. Experiments to investigate further the effects of dexamethasone on the expression of StAR mRNA stimulated with LH and cAMP are currently in progress. In addition, future studies should be directed toward elucidation of the mechanism(s) by which glucocorticoids modulate StAR gene expression and identification of transcription factors that mediate both cAMP- and glucocorticoid-induced modulation of expression. Because StAR is transcriptionally regulated by the orphan nuclear receptor, steroidogenic factor 1 (SF-1) [54], dexamethasone-induced inhibition of StAR may occur via the same mechanism involving a common regulator like SF-1. DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome, gene 1), which also belongs to the orphan nuclear receptor family and has been found to inhibit SF-1 transactivation [55], is another potential target of dexamethasone action.

Dexamethasone has also been shown to inhibit cAMP-induced synthesis of P450scc enzyme in mouse Leydig cells by a receptor-mediated mechanism [56], whereas in Leydig tumor cells, dexamethasone potentiated cAMP-induced synthesis of P450scc enzyme [57]. To date, the reason for the different glucocorticoid actions in normal and neoplastic cells is not fully understood. However, in our study, the amount of P450scc enzyme, as determined by Western analysis, was not changed by the dexamethasone treatment. The differences in results may be due to tissue specificity. Our results further suggest that the decreased steroidogenic response of preovulatory follicles to dexamethasone may be essentially localized to the transfer of cholesterol to the inner mitochondrial membrane. Whether dexamethasone treatment affects the activity of P450scc enzyme in preovulatory follicles remains to be investigated.

In conclusion, the results of the present study indicate that in rat preovulatory follicles, dexamethasone acts to repress the LH-induced expression of StAR protein and progesterone production by a glucocorticoid receptor-mediated mechanism. These observations raise the possibility that glucocorticoids in vivo may act directly on the ovary to modulate follicular steroidogenesis. Additional studies are necessary to clarify the mechanism(s) involved in the suppressing effect on the expression of StAR protein.

ACKNOWLEDGMENTS

We thank Dr. Douglas M. Stocco, Department of Cell Biology and Biochemistry, School of Medicine, Texas Tech University, Health Science Center for his supply of antiserum for the StAR protein and Dr. Bon-Chu Chung, Institute of Molecular Biology, Academia Sinica for her supply of P450scc antibody. Special thanks are also given to Mr. Jyh-Chen Lin for his technical assistance in progesterone RIA.

FOOTNOTES

First decision: 7 July 2000.

1 This study was supported by a grant (NSC88-2314-B006-069) from the National Science Council, Republic of China. Back

2 Correspondence. FAX: 886 6 236 2780; lifupi{at}mail.ncku.edu.tw Back

Accepted: August 15, 2000.

Received: June 13, 2000.

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