Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 66, 959-965 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effects of Progestins on Progesterone Synthesis in a Stable Porcine Granulosa Cell Line: Control of Transcriptional Activity of the Cytochrome P450 Side-Chain Cleavage Gene1

C.L. Swana, M.C. Agostinib, P.M. Bartlewskia, V. Feyles3,,a, R.J. Urbanc, and P.J. Chedrese2,,a

a Department of Obstetrics, Gynecology & Reproductive Sciences, University of Saskatchewan,Saskatoon, Saskatchewan, Canada S7N OW8 b Facultad de Ciencias Veterinarias, UNCPBA, Tandil, Argentina c Department of Internal Medicine/Endocrinology, The University of Texas Medical Branch, Galveston, Texas 77555-1060


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of the present study was to examine the effects of progestins on progesterone synthesis and expression of the cytochrome P450 cholesterol side-chain cleavage gene (P450scc) in a stable porcine granulosa cell line, the JC-410. Cells were incubated for 48 h with the synthetic progestogen-levornorgestrel with or without RU486 (progesterone and glucocorticoid receptor antagonist) or RWJ26819 (progesterone agonist without affinity to glucocorticoid receptors). Both levonorgestrel and RU486 enhanced progesterone accumulation in a dose-dependent manner. RU486 did not antagonize the effects of levonorgestrel, and RWJ26819 had no effect on progesterone production in cultured JC-410 cells. Progesterone and levonorgestrel increased steady state P450scc mRNA levels after 3–6 h of treatment. Progesterone and RU486 at 0.1, 1, and 10 µM increased the transcription rate of P450scc transiently expressed in JC-410 cells after 18 h of incubation; 30 µM had no effect, and 100 µM suppressed transcription. Levonorgestrel did not affect transcription of the P450scc gene, and RWJ26819 reduced its transcription. Progesterone and RU486 significantly decreased the number of cells and total protein content after 72 and 24 h of incubation, respectively. Levonorgestrel had no effect, whereas RWJ26819 increased (24 h) but subsequently reduced (72 h) cell number and protein content. The present results indicate that progestins are capable of directly modulating progesterone biosynthesis in porcine JC-410 granulosa cells. These effects may be exerted in part through the regulation of P450scc gene expression. Ostensible differences exist between progesterone and its synthetic analogues in the control of progesterone secretion in the stable porcine granulosa cell line in vitro.

follicle, granulosa cells, mechanisms of hormone action, ovary, steroid hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Progesterone is a steroid hormone produced in the ovary under the control of the pituitary gonadotropins [1]. The corpus luteum, which forms from the remnants of an ovulated follicle, is a major source of the total circulating progesterone during the luteal phase of the estrous/menstrual cycle [2]. Granulosa and theca cells are the primary sites of progesterone biosynthesis during follicular growth and differentiation [1]. Progesterone has endocrine actions in several organs, including the uterus, mammary gland, brain, and bones [1]. It has also been suggested that progesterone may have direct effects on follicular development, ovulation, and luteogenesis in a number of mammalian species [3–8]. From earlier studies performed mainly in rodents, it is plausible that locally produced steroids exert autocrine, paracrine, and endocrine effects on ovarian steroidogenesis [5, 7, 9, 10]. In 1981, Rothchild [9] proposed that progesterone stimulated its own synthesis and hence played a pivotal role in the control of corpus luteum function. Results of more recent studies are supportive of this notion [11–16]. Similar information for other species is still limited, and there is a paucity of information on the local effects of progestins in ovarian follicular steroidogenesis and granulosa cell function [7]. We have recently demonstrated that the synthetic progestogen, levonorgestrel, increases progesterone accumulation in cultured, stable porcine granulosa cells, the JC-410 [17, 18]. Results of those studies have been interpreted to suggest that progestins may affect progesterone synthesis by the regulation of steroidogenic enzymes, the cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD). However, the molecular mechanism or mechanisms through which progestins exert these effects are not completely understood.

There is increasing evidence that progestins may directly modulate progesterone synthesis in the luteinizing antral follicle and corpus luteum, but similar studies for the granulosa cells from growing antral follicles are scarce. Furthermore, previous in vitro studies of the role of progestins in ovarian steroidogenesis have been commonly conducted in primary cultures of granulosa or luteal cells. These cells release large amounts of steroid hormones, the accumulation of which can sometimes mask the effects produced by hormonal stimulation [19, 20]. JC-410 cells secrete relatively low levels of progesterone and respond to stimulation with steroid hormones and with activators of the protein kinase A (PKA) pathway in the same manner as primary cultures of granulosa cells [17]. Moreover, JC-410 cells appear to be arrested in an undifferentiated stage of development, in which the gonadotropin receptors are not expressed [21]. Response to FSH cannot be restored in cultured JC-410 cells even after treatments with agents that typically induce FSH receptor responsiveness, such as activators of the PKA pathway, estrogens and activin [21]. Therefore, the JC-410 cell line provides a useful experimental model for studying the direct effects of steroid hormones on progesterone synthesis in vitro.

It has been suggested in rodents that the actions of progestins in the ovary may be mediated through classical progesterone receptors as well as glucocorticoid receptors [22]. Hence, the objective of this study was to examine the effects of progesterone, levonorgestrel (a synthetic progestogen), RU486 (a potent antiprogestin and antiglucocorticoid), and RWJ26819 (a progestin agonist without affinity to the glucocorticoid receptor) on the regulation of progesterone synthesis in JC-410 cells. Of particular interest were the effects of progesterone and its analogues or antagonists on the expression of the cytochrome-P450scc gene, a rate-limiting step in steroidogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Reagents for tissue cultures and Lipofectamine-2000 were purchased from Gibco (Burlington, ON, Canada). Bovine insulin, cholera toxin, and progesterone (4-pregnene-3,20-dione) were purchased from Sigma Chemical Company (St. Louis, MO). Iodinated progesterone (11ß-hydroxyprogesterone-11-D-glucuronide-125I-iodotiramine) and Hybond nylon membranes were purchased from Amersham Pharmacia Biotech (Baie d'Urfe, PQ, Canada). 32P was purchased from New England Nuclear (Boston, MA). The Bio-Rad DC Protein Assay kit was purchased from Bio-Rad Laboratories (Hercules, CA). Plastic culture plates were purchased from Falcon (Lincoln Park, NJ). Levonorgestrel (13-ethyl-17-ethynyl-17-hydroxygen-4-en-3-one) was provided by Wyeth-Ayerst (Montreal, PQ, Canada). RU486 was provided by Exelgyn Laboratories (Paris, France). RWJ26819 was a generous gift from Johnson Pharmaceutical Research Institute (Raritan, NJ).

Cell Culture

The immortalized porcine granulosa cell line, JC-410, was cultured as described previously [20]. Briefly, cells were grown in Media 199 supplemented with 5% newborn calf serum (NBCS), 5 µg/ml insulin, 100 IU/ml penicillin, and 100 µg/ml streptomycin. All experiments were performed at 70%–90% cell confluency, in serum-free culture media.

Progesterone and Protein Assays

Progesterone content was determined by radioimmunoassay (RIA) in 100 µl of culture medium, as described elsewhere [20]. The lowest detectable concentration of progesterone was 6.25 pg, and the intraassay and interassay coefficients of variation (CVs) were <10%. Both levonorgestrel and RU486 have been found to cross-react with the progesterone antibody. Therefore, cross-reactivity was subtracted from the progesterone concentrations. After incubation, cells were extracted with 0.1% sodium dodecyl sulfate, and protein concentrations were measured using the Bio-Rad DC Protein Assay kit. Finally, progesterone concentrations determined by RIA were normalized between experiments by dividing the progesterone levels measured by their respective cellular protein contents. Results were expressed as fold increases or decreases above or below untreated controls.

Determination of Cell Viability

Cells were plated in 24-well plates for up to 72 h. At the end of incubation, the wells were washed with 0.9% PBS solution, and all remaining cells attached to the plate were regarded as viable cells. Such cells were then recovered with 100 µl of 0.25% trypsin. Cell suspensions were pelleted and subsequently resuspended in 30 µl of culture media containing 15 µl of trypan blue for enumeration using a hemocytometer.

Northern Blot Analyses

Total RNA was isolated by an acid-phenol-chloroform extraction according to the method described by Chomczynski and Sacchi [23]. RNA was denatured, then size-fractionated by electrophoresis on 1% agarose-formaldehyde gel (10 µg RNA per lane), and transferred onto a nylon membrane by diffusion blotting. Transferred RNA was cross-linked to the membrane using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Complementary DNA for porcine P450scc [24] and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; [25]) were used as probes. Complementary DNA was labeled by primer extension [26] with a [32P]dCTP (>3000 Ci/mmol; New England Nuclear) to a final specific activity of 1.5–3.0 x 109 disintegrations per minute/mg DNA. Membranes were then hybridized and autoradiographed as previously described [27].

Transfection of JC-410 Cells with the 2320-P450scc-LUC Construct

The reporter gene construct, 2320-P450scc-LUC, has been previously described [28]. Briefly, the construct contains the entire sequenced promoter region of the porcine P450scc gene linked to the complete coding region of the firefly luciferase (LUC) gene, including the polyadenylation signal. Optimal conditions for transient expression of the construct were based on preliminary trials, in which the relative changes in the rates of basal transcriptional activity over time were determined. The JC-410 cells were cultured in 24-well plates and transfected with 1 µg DNA/well using Lipofectamine-2000 reagent in Media 199 (500 µl) containing 5% NBCS and without antibiotics (transfection mixture). After 24 h, media were replaced with fresh media containing the hormonal treatments, and incubated for 18 h. Experiments were terminated by washing cell cultures with 0.9% PBS, and cells were collected for luciferase assay.

Luciferase Assay

Cultured cells were harvested by adding 200 µl/well of extraction buffer (1% Triton X-100 and 1 mM dithiothreitol [DTT]). Cell extracts were centrifuged for 5 min at 4°C (12 000 x g). Luciferase activity was measured in 100 µl of the supernatant diluted in 368 µl of luciferase buffer (25 mM gly-glycine, 15 mM magnesium sulphate, 4 mM EGTA, 16 mM potassium phosphate, 1 mM DTT, and 2 mM ATP pH 7.8) at room temperature. Light emission was determined using a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA), which injects 100 µl of 25 mM luciferin in glyclglycine buffer (25 mM glyclglycine, 15 mM magnesium sulphate, 4 mM EGTA, and 10 mM DTT pH 7.8) into the cell lysate in the luciferase buffer. Luciferase activity was measured by calculating the light emitted during the initial 10 sec of reaction, and the values were expressed as arbitrary light units. The results were then normalized by dividing the values for each treatment by the respective control values (i.e., readings for untreated control cells exposed to the transfection mixture), and expressed as fold increases or decreases over the mean control level.

Statistical Analyses

All results are given as means ± SEM of three independent replicates. Initially, data were subjected to two-way ANOVA (SigmaStat Statistical Software, version 2.0 for Windows 95, NT, and 3.1, 1997; Chicago, IL) to assess the main effects of replicate (1–3), treatment, and the interaction of these terms. If the main effects of replicate and interaction terms were not significant (P > 0.05), data from all replicates were pooled and analyzed by one-way ANOVA. The Fisher protected least significant difference [29] was used as a post-ANOVA test for comparisons of individual means.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Progestins and RU486 on the Viability of JC-410 Cells

The effects of progesterone, levonorgestrel, RU486, and RWJ26819 on the viability and protein content of cultured JC-410 cells are illustrated in Figure 1. Within experiments, there were numerical but not significant fluctuations in the total cell number and protein content of control (nonstimulated) JC-410 cells maintained in serum-free media for up to 72 h. Progesterone, at 10 µM, decreased the total number of cells and protein content after 72 h of incubation. RU486 at 10 µM significantly reduced cell number and protein concentrations after 24, 48, and 72 h. Levonorgestrel (10 µM) had no effect on cell viability up to 72 h of incubation (P > 0.05). Treatment with 10 µM RWJ26819 increased (24 h) and then decreased (72 h) the total cell numbers and protein content (P < 0.05).



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  		FIG. 1. Effects of progesterone (P4), levonorgestrel (LNG), RU486 (Mifepristone), and RWJ26819 on the viability (A) and protein content (B) of JC-410 cells in culture. Cells were incubated for up to 72 h in the absence (controls) or presence of 10 µM P4, LNG, RU486, or RWJ26819. Bars represent means ± SEM of three independent experiments. Values are expressed as a fold over mean control value for each treatment. Asterisks denote values that were significantly different from respective controls (P < 0.05)

Effects of Levonorgestrel on Progesterone Synthesis

Mean concentrations of progesterone in culture media after incubation of the JC-410 cells with increasing concentrations of levonorgestrel are given in Figure 2A. Levonorgestrel at 3, 10, and 30 µM increased (P < 0.05) progesterone synthesis by 1.9-, 4.0-, and 3.1-fold, respectively, after 48 h. Maximal stimulation was obtained with 10 µM levonorgestrel (P < 0.05), and so this concentration was used in subsequent experiments.



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  		FIG. 2. A) Mean (± SEM) concentrations of progesterone in culture media after incubation of JC-410 cells with control media or increasing amounts of levonorgestrel (LNG; 0.3, 1, 3, 10, and 30 µM) for 48 h. B) Effect of time on LNG-stimulated progesterone accumulation; JC-410 cells were incubated in the presence or absence of 10 µM LNG for 3, 6, 12, 24, and 48 h. Means denoted by different letters are different (P < 0.05)

The effect of duration of the treatment with levonorgestrel (10 µM) on progesterone synthesis by the JC-410 cells is illustrated in Figure 2B. Levonorgestrel increased (P < 0.05) progesterone synthesis by 2.0-, 2.5-, 2.8-, 3.4-, and 5.0-fold (fold increases over mean control levels) after 3, 6, 12, 24, and 48 h of incubation, respectively. Both basal (nonstimulated) and levonorgestrel-induced progesterone accumulation rose (P < 0.05) between 6 and 12 h, and again between 24 and 48 h; there were no differences (P > 0.05) from 12 to 24 h of the incubation period.

Effects of RU486 and RWJ26819 on Levonorgestrel-Stimulated Progesterone Synthesis

RU486 at 10 and 100 µM increased (P < 0.001) progesterone synthesis by JC-410 cells after 48 h by 4.3- and 10.4-fold, respectively (Fig. 3). However, treatment with 100 µM RU486 caused the cells to detach from the plate, indicating increased cell death, and RU486 was seen to precipitate in the culture media.



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  		FIG. 3. Effect of RU486 (Mifepristone) on progesterone synthesis in cultured JC-410 cells. Cells were incubated with control media, 0.1, 1, 10, or 100 µM RU486 for 48 h. Each bar represents the mean ± SEM of three independent experiments. Values are expressed as a fold over mean control value for each treatment. Means denoted by different letters are different (P < 0.05).

Mean concentrations of progesterone following the 48-h treatment of JC-410 cells with control media or 10 µM levonorgestrel, in the presence or absence of 10 µM RU486, are shown in Figure 4. Levonorgestrel and RU486 caused 3.3- and 1.9-fold increases in progesterone synthesis, respectively (P < 0.05). Treatment with levonorgestrel and RU486 resulted in a 4.4-fold increase in progesterone concentrations; the combined effect of the two compounds was greater than that produced by levonorgestrel and RU486 alone (Fig. 4A; P < 0.05). There were no differences (P > 0.05) in progesterone synthesis between the JC-410 cells incubated for 48 h with levonorgestrel alone or levonorgestrel and RWJ26819, and no significant effect of RWJ26819 on progesterone accumulation (Fig. 4B). Levonorgestrel (10 µM) with or without 30 µM RWJ26819 increased progesterone synthesis by 3.0-fold (P < 0.001).



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  		FIG. 4. Effects of RU486 (A) and RWJ26481 (B) on levonorgestrel (LNG)-stimulated progesterone accumulation in cultured JC-410 cells. Cells were incubated with control media or 10 µM LNG in the presence or absence of 10 µM RU486 or 30 µM RWJ26819 for approximately 48 h; RU486 and RWJ26819 were added 1 h prior to addition of LNG. Each bar represents mean ± SEM of three independent experiments. Values are expressed as a fold over mean control value for each treatment. Different letters indicate significant differences (P < 0.05).

Effect of Time on Progestin-Stimulated P450scc mRNA Steady-State Levels

The effect of time on progesterone and levonorgestrel-stimulated P450scc mRNA levels is shown in Figure 5. There were no significant differences in steady-state levels of P450scc mRNA in the JC-410 cells cultured from 12 to 48 h before the treatment (control cells). P450scc mRNA levels increased approximately 2.0-fold relative to the control value at 3 h, to 2.5-fold at 6 h, and subsequently returned to control levels after 12 h of exposure, for both progesterone and levonorgestrel treatments (10 µM). The JC-410 cells were also incubated for 24 h with 100 ng/ml of cholera toxin (positive control). In all experiments, cholera toxin increased P450scc mRNA levels (range, 3.0- to 14.0-fold; P < 0.05; data not shown).



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  		FIG. 5. Effect of time on progesterone (P4) and levonorgestrel (LNG)-stimulated steady-state P450scc mRNA levels. Total mRNA was extracted and analyzed by Northern blot hybridization and GAPDH was used as a control for RNA loading. A) An autoradiograph of a representative experiment; B) a graph summarizing the results of densitometric analysis of an autoradiograph shown in A. Values graphed are the ratios of P450scc:GAPDH and are expressed as fold increases over a control value (Time 0)

Effects of Progestins and RU486 on Transcriptional Activity of the P450scc Gene

The relative transcription rates of the P450scc gene, as determined by a luminometric assay following the transfection of 2320-P450scc-LUC, are presented in Figure 6. Progesterone at 0.1, 1, and 10 µM increased (P < 0.05) transcriptional activity of the 2320-P450scc-LUC by 1.7-, 1.8-, and 2.0-fold, respectively; 30 µM had no effect (0.9-fold; P > 0.05), and 100 µM completely suppressed transcription (0.03-fold; P < 0.001). Levonorgestrel, over the entire range of concentrations studied (0.1, 1, 10, 30, and 100 µM), did not affect (P > 0.05) the transcription of the 2320-P450scc-LUC. However, treatment with 0.1 µM levonorgestrel tended to inhibit the transcription (0.6-fold; P = 0.053). RU486 at 1 and 10 µM enhanced the rate of transcription of the 2320-P450scc-LUC by 1.7- and 1.6-fold, respectively (P < 0.05), whereas 100 µM produced a decline to a 0.4-fold (P < 0.05). RWJ26819 significantly depressed the transcription of the P450scc gene (0.5-, 0.6-, 0.6-, 0.6-, and 0.4-fold, for 0.1, 1, 10, 30, and 100 µM RU26819, respectively). In addition, the effect of levonorgestrel and RWJ26819 on the transcriptional activity of 2320-P450scc-LUC was lower compared to that of progesterone and RU486 at 0.1, 1, and 10 µM. However, it was lower for progesterone, RU486, and RWJ26819 compared to levonorgestrel, at 30 and 100 µM (P < 0.05).



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  		FIG. 6. Effects of progesterone (P4), levonorgestrel (LNG), RU486 (Mifepristone), and RWJ26819 on transcriptional activity of the P450scc gene transiently expressed in the JC-410 cells. The results (means ± SEM) are compiled from three independent experiments, and values are expressed as a fold over mean control value for each replicate. Asterisks denote significant differences relative to respective untreated controls: large asterisk, P < 0.05; small asterisk, P < 0.10. See text for additional statistical descriptions


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have suggested that progesterone may have several intraovarian actions, including the control of folliculogenesis, steroidogenesis, ovulation, and formation of the corpus luteum [5]. Rothchild [9] proposed that one of the primary functions of progesterone in the ovary might be the regulation of its own synthesis. The results of the present study clearly indicate that levonorgestrel stimulated progesterone synthesis in the porcine granulosa cells in vitro in a time- and dose-dependent fashion (Fig. 2). The stimulatory effects of levonorgestrel on progesterone accumulation in JC-410 cells occurred independently of gonadotropic hormones; JC-410 cells do not respond to gonadotropins, hence the effects of steroids seen were independent of gonadotropic influences. It has been shown that the synthetic progestins, such as R5020 and medroxyprogesterone acetate, augment gonadotropin-stimulated progesterone synthesis in rat granulosa cells [30–32]. Progesterone synthesis in a human granulosa/lutein cell line in vitro cannot be restored by hCG stimulation after RU486 treatment [33]. Collectively, these observations suggest that progestins may modulate progesterone synthesis in a gonadotropin-independent manner, or that the presence of progesterone/progestins may be essential for gonadotropin-induced progesterone synthesis in the granulosa cells.

In the present study, the anti-progestin RU486 increased progesterone synthesis when administered alone or in combination with levonorgestrel (Figs. 3 and 4). An increase in progesterone synthesis after treatment with RU486 was all the more pronounced considering that RU486 significantly reduced the number and protein content of the JC-410 cells after 24 h of incubation (Fig. 1). The present results are in accordance with earlier observations that RU486 could act as a progestin [15, 34]. In postmenopausal women, RU486 in the absence of progesterone has a progestogenic effect [34]. The granulosa cells obtained from ovarian follicles before the LH surge also respond to RU486 with an increase in progesterone synthesis [5]. It is therefore attractive to speculate that under diminished gonadotropic and progestogenic stimulation, RU486 can act as a progesterone agonist in the ovarian cells.

It is possible that the diverse effects of RU486 on progesterone synthesis are associated with changes in populations of progesterone receptor (PR) isoforms. Such a possibility has been previously postulated by Rothchild [10]. The PR isoforms, A and B, have been shown to have opposing function in primates [35]. The activation of the B isoform of PR always exerts agonistic effects, even after binding to a progesterone antagonist such as RU486, whereas the A isoform always acts as an antagonist [35]. In most tissues, PR-A and PR-B coexist in equimolar amounts. However, a significant increase in total PR population is observed after the preovulatory LH surge and just before ovulation in primates' ovarian follicles [5]. This increase in the expression of PRs coincides with the shift in RU486 activity from a progestin agonist to antagonist [5]. A possible explanation for this change is that the LH discharge stimulates an increase in PR-A expression, which would antagonize the effects of RU486 bound to PR-B. This leads us to hypothesize that the stimulatory effects of RU486 may be due to the presence of higher levels of PR-B. JC-410 cells are derived from granulosa cells of prepubertal gilts, whose ovaries had not been exposed to follicular-phase levels of LH. Therefore, the ratio of PR isoforms in JC-410 cells could resemble that in primary cultures of granulosa cells, in which RU486 has agonistic effects (i.e., granulosa cells obtained before the LH surge [5]). The presence of the two isoforms of PR has been confirmed in the granulosa cells of early antral and preovulatory follicles in the pig [36]. In addition, stimulation with LH and FSH increases the population of PRs in porcine granulosa cells in vivo [36] and enhances PR gene expression in vitro [37].

The effects of RU486 on progesterone accumulation could have been elicited through the interaction with other steroid hormone receptors. Sugino et al. [22] demonstrated that progesterone decreased 20{alpha}-hydroxysteroid dehydrogenase (20{alpha}-HSD) activity in the rat corpus luteum by interacting with glucocorticoid receptors. RU486 is a potent antiglucocorticoid that binds with high affinity to the glucocorticoid receptor. In addition, RWJ26819 that does not bind to the glucocorticoid receptor failed to increase progesterone synthesis in this study. We suggest that the stimulatory effects of RU486 and progestins on progesterone synthesis could be mediated, at least in part, via the glucocorticoid receptor.

The presence of progesterone and glucocorticoid receptors in the JC-410 cell line has yet to be established. Future studies aimed at characterizing progesterone and glucocorticoid receptor expression in JC-410 cells will be necessary to determine the complete mechanism of action of these steroids in the granulosa cells obtained from antral follicles of pigs. Whether steroid hormones may exert direct modulatory effects on follicular steroidogenesis in the presence of gonadotropic stimulation (i.e., in the porcine granulosa cells expressing LH/FSH receptors) also remains to be elucidated.

Progesterone and levonorgestrel (10 µM) increased the steady state levels of P450scc mRNA in JC-410 cells (Fig. 5). The results of Northern blot analyses suggest that regulation of P450scc gene expression by progestins is time dependent. The stimulatory effect was observed at 3–6 h of exposure and was followed by a decline in mRNA levels at 12 h. In an earlier study [38], progesterone decreased P450scc activity in the rat corpus luteum after 9 days. It is, however, important to note that changes in mRNA levels do not always reflect the alterations in enzyme activity. There was also a discrepancy between the present Northern blot analysis and assessment of transcriptional activity of P450scc by a luciferase assay. Progesterone (0.1, 1, and 10 µM) and RU486 (1 and 10 µM) significantly increased the transcription of P450scc following the 18-h treatment (Fig. 6). Higher concentrations of progesterone and RU486 had no effect or inhibited the transcription of the P450scc gene (Fig. 6). It is interesting that levonorgestrel, over the entire range of concentrations studied, did not significantly affect the transcription of P450scc, and RWJ26819 inhibited transcription (Fig. 6). The results of Northern blot analysis and luciferase assays are intriguing, and suggest that prolonged exposure to progesterone and RU486, and elevated concentrations of these, may suppress P450scc gene expression in granulosa cells. Hence, the effects of progestins on the expression of the P450scc gene appear to be both time- and dose-dependent, with an initial stimulation followed by an inhibition. However, in light of the present results, the local effects of levonorgestrel on progesterone synthesis cannot be explained solely by the control of P450scc gene transcription. Levonorgestrel-stimulated progesterone secretion may be due mainly to the stimulation of 3ß-HSD gene expression [18] or regulation of other steps in the biosynthesis of progesterone (i.e., expression of steroid acute regulatory protein [39]).

In 1994, Telleria and Deis [15] demonstrated that RU486 could both stimulate and inhibit ovarian steroidogenesis in vivo. The present study is the first demonstration of the dual effect of RU486 and progesterone on transcriptional activity of the P450scc gene in granulosa cells in vitro. In this study, we were unable to detect a decline in progesterone levels after treatment with RU486 at the concentrations that suppressed P450scc gene transcription. This is probably because an initial stimulation of the P450scc gene expression, allowing for a transient increase in progesterone synthesis, preceded a suppression of transcription. In this model, progesterone accumulation over time is measured. This precluded our being able to detect an inhibition of progesterone synthesis and the time at which it occurred. However, a decline in P450scc expression, a rate-limiting step in steroidogenesis, inevitably causes a reduction in progesterone synthesis. We propose that the paradoxical effect of RU486 on progesterone synthesis may be due to an initial stimulation followed by an inhibition of P450scc gene transcription.

In summary, levonorgestrel and RU486, but not RWJ26819, stimulated progesterone synthesis in porcine JC-410 granulosa cells in a gonadotropin-independent manner. The rise in progesterone synthesis appears to be mediated at least in part through increased expression of the P450scc gene. Levonorgestrel appeared to be considerably less potent than progesterone and RU486 in terms of the stimulation of the P450scc gene transcription. We conclude that progestins can modulate progesterone synthesis in JC-410 porcine granulosa cells in vitro. The present results are also indicative of differences in one or more mechanisms by which progesterone and synthetic progestogens modulate progesterone secretion in cultured porcine granulosa cells.


   ACKNOWLEDGMENTS
 
We thank Dr. Steve Palmer and the R.W. Johnson Pharmaceutical Research Institute (Raritan, NJ) for the provision of RWJ26819 and Dr. Regine Sitruk-Ware of the Exelgyn Laboratories (Paris, France) for provision of RU486.


   FOOTNOTES
 
First decision: 19 July 2001.

1 Financial support of the College of Graduate Studies and Research, University of Saskatchewan to C.L.S. is gratefully acknowledged. M.C.A. was a recipient of a UNCPBA Program 7 postgraduate fellowship. P.M.B. was a recipient of a Health Services Utilization and Research Commission (HSURC) postdoctoral fellowship. This study was funded by the Canadian Network of Toxicology Centers, Saskatchewan Health (Government of Saskatchewan)/Canadian Institute for Health Research Regional Partnership, National Sciences and Engineering Research Council of Canada, and Saskatchewan Health Services Utilization and Research Commission. Back

2 Correspondence: P. Jorge Chedrese, Department of Obstetrics, Gynecology & Reproductive Sciences, Royal University Hospital, 103 Hospital Dr., Saskatoon, SK, Canada S7N OW8. FAX: 306 966 8040; jorge.chedrese{at}usask.ca Back

3 Current address: Department of Obstetrics and Gynecology, Reproductive Endocrinology and Infertility Program, London Health Sciences Center, University of Western Ontario, London, ON, Canada N6A 5A5 Back

Accepted: November 1, 2001.

Received: June 21, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Graham JD, Clarke CL. Physiological actions of progesterone in target tissues. Endocr Rev 1997; 18:502-519[Abstract/Free Full Text]
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