Molecular Human Reproduction, Vol. 6, No. 3, 238-245,
March 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Involvement of progesterone in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide gene expression in pre-ovulatory follicles of rat ovary
1 Hormone Research Center, 2 Department of Biology, Chonnam National University, Kwangju 500–757, Republic of Korea, and 3 Department of Biological Regulation, Bernhard Zondek Hormone Research Laboratory, Weizmann Institute of Science, Rehovot 76100, Israel
Abstract
The present study was designed to determine whether progesterone might have a role in gonadotrophin-induced pituitary adenylate cyclase-activating polypeptide (Pacap) gene expression in rat ovary. Northern blot analysis revealed that treatment of pregnant mare's serum gonadotrophin (PMSG)-primed immature rats with the progestin antagonist RU486 or an inhibitor of 3ß-hydroxysteroid dehydrogenase epostane, 1 h before HCG, resulted in a dose-dependent inhibition of the HCG-induced Pacap gene expression. In-situ hybridization demonstrated that the number of pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells was greatly reduced in ovaries treated with RU486. Moreover, the suppressive effect of RU486 or epostane on the LH-induced Pacap gene expression in cultured pre-ovulatory follicles was reversed by co-treatment with the synthetic progestin R5020. We further cloned the 5'-flanking region of the rat Pacap gene and identified the presence of a consensus progesterone receptor element. When luciferase fusion genes containing Pacap gene promoter were transiently transfected into granulosa cells of pre-ovulatory follicles, luciferase activity was markedly stimulated by LH. Treatment with RU486 or epostane resulted in partial suppression of LH-stimulated PACAP promoter activity. Taken together, these results indicate that progesterone, acting through progesterone receptors, plays a role in gonadotrophin induction of Pacap gene expression in granulosa cells of pre-ovulatory follicles, and thereby may be involved in the process of ovulation.
gonadotrophin/ovary/ovulation/Pacap/progesterone receptor
Introduction
The pre-ovulatory surge of LH is obligatory to trigger ovulation, a process by which pre-ovulatory follicles rupture, release a fertilizable oocyte and undergo luteinization. Several studies have shown that the LH surge induces the rapid, but transient, expression of specific genes associated with ovulation in granulosa cells of pre-ovulatory follicles (Richards, 1994
). These specific genes induced by LH include the progesterone receptor (PR) which is a member of the nuclear receptor superfamily of transcription factors (Vegeto et al., 1993) and pituitary adenylate cyclase-activating polypeptide (PACAP), a novel neuropeptide with considerable homology to vasoactive intestinal peptide and growth hormone releasing hormone (Arimura, 1992).
It has been shown that PR mRNA is transiently induced in granulosa cells of pre-ovulatory follicles by gonadotrophins in the rat ovary (Park and Mayo, 1991; Natraj and Richards, 1993). Several lines of evidence indicate that gonadotrophins activate the PR gene through a cAMP stimulation of the A-kinase pathway (Park-Sarge and Mayo, 1994; Park-Sarge and Sarge, 1995). Furthermore, recent study demonstrates that the phosphorylation of some transcription factors, in addition to the oestrogen receptor, by gonadotrophin-activated the A-kinase pathway is critical for the transactivation of the PR gene (Clemens et al., 1998). The induction of PR by LH has been suggested to be critical for progesterone action in regulating ovulation and activating specific genes. Inhibitors of progesterone biosynthesis and antiprogestin can block LH induction of ovulation in vivo (Tsafriri et al., 1987; Tanaka et al., 1992; Uilenbroek et al., 1992). PR has also been shown to play a functional role in the process of luteinization in vitro (Natraj and Richards, 1993). Furthermore, a targeted deletion of the PR gene in mice causes the failure to ovulate even when exogenous gonadotrophins are administered (Lydon et al., 1995), confirming a key role for PR in the LH-induced process of ovulation. However, target genes for PR action in the ovary have not yet been identified.
We have recently shown that gonadotrophins also induce the rapid and transient expression of Pacap mRNA in granulosa cells of pre-ovulatory follicles by activating a cAMP-mediated pathway (Lee et al., 1999). Interestingly, the expression of Pacap mRNA appears to be delayed a few hours compared with that of PR mRNA after gonadotrophin stimulation (Park and Mayo, 1991; Natraj and Richards, 1993; Lee et al., 1999), implying that PR may regulate Pacap gene expression. Furthermore, a recent report demonstrates that PACAP plays a role in peri-ovulatory progesterone production and subsequent luteinization in rat granulosa/lutein cells (Gräs et al., 1999). Thus, it appears plausible that one of the possible target genes for PR action in the ovary might be a PACAP. Based on these considerations, we have sought to determine whether progesterone, acting through PR, plays a role in gonadotrophin stimulation of Pacap gene expression in rat pre-ovulatory follicles. The present study shows that RU486, an antiprogestin, and epostane, an inhibitor of 3ß-hydroxysteroid dehydrogenase, could block LH/HCG-induced Pacap gene expression in granulosa cells of pre-ovulatory follicles both in vivo and in vitro. We have also cloned the promoter region of the rat Pacap gene and examined the effect of RU486 or epostane on LH-stimulated PACAP promoter activity in transfected granulosa cells.
Materials and methods
Hormones and reagents
Ovine LH (LH-S-26; 2 300 IU/mg) was obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD). HCG and pregnant mare's serum gonadotrophin (PMSG) were purchased from Sigma Chemical Co (St Louis, MO, USA). The progestin antagonist mifepristone (RU486) was purchased from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA, USA). Inhibitor of 3ß-hydroxysteroid dehydrogenase, epostane (2
,4,17–4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxoandrostane-2-carbonitrile), was provided by the courtesy of Dr B.W.Snyder (Sterling-Winthrop, New York, NY, USA). The synthetic progestin R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20 dione) was the generous gift of Dr J.P.Raynaud (Roussel-UCLAF, Romainville, France).
Animals
Immature female rats of the Sprague–Dawley strain were purchased from Daehan Laboratories (Chungbuk, Korea). They were housed in groups in a room with controlled temperature and photoperiod (10 h darkness/14 h light, with lights on from 06:00–20:00 h). The animals had access to food and water ad libitum. At 26 days old, the animals (body weight, 60–65 g) were injected s.c. with 10 IU PMSG to induce multiple follicle growth. After 48–52 h, the animals were killed by cervical dislocation and the ovaries were removed for follicle dissection or granulosa cell collection. Some rats received i.p. administration of RU486 or epostane, 1 h before 10 IU HCG injection to induce ovulation, and ovaries were obtained 6 h after stimulation for Northern blot and in situ hybridization analysis.
Northern blot analysis
Total RNA from ovaries or cultured follicles was isolated using Tri Reagent solution (Sigma Chemical Co). Total RNA (10–20 µg) were fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and were transferred to nylon membranes by capillary blotting with 10x sodium citrate/sodium chloride (SSC). After UV cross-linking and prehybridization, membranes were hybridized overnight at 42°C in a solution containing 50% formamide, 5x SSC, 1 mmol/l EDTA, 250 µg/ml denatured salmon sperm DNA, 500 µg/ml yeast transfer RNA, and a total of 2–4x106 cpm of a [32P]-labelled full-length rat Pacap complementary DNA (cDNA) probe (Lee et al., 1999). After hybridization, membranes were washed twice for 5 min at room temperature in 2x SSC and 0.1 sodium dodecyl sulphate (SDS), followed by 1 h at 65°C in 0.5x SSC and 0.1% SDS. Membranes were then exposed using Kodak RX films (Eastman Kodak Co, Rochester, NY, USA) for 1–3 days at –80°C. The signals were normalized to the 28S ribosomal RNA internal control.
In-situ hybridization analysis
Ovaries were fixed at 4°C for 6 h in 4% paraformaldehyde in PBS, followed by immersion in 0.5 M sucrose in PBS overnight. Cryostat sections (14-µm thick) were mounted on poly-L-lysine (Sigma Chemical Co)-coated microscope slides, fixed in 4% paraformaldehyde in PBS, and stored at –80°C until analysed. The hybridization procedure was essentially the same as previously described (Lee et al., 1999). In brief, sections were pretreated serially with 0.2 mol/l HCl, 2x SSC, pronase (0.125 mg/ml), 4% paraformaldehyde, and acetic anhydride in triethanolamine. Hybridization was carried out at 52–55°C overnight in the mixture containing [35S]-labelled rat Pacap cRNA probe (108 cpm/ml), 50% formamide, 0.3 mol/l NaCl, 10 mmol/l Tris–HCl, 5 mmol/l EDTA, 1x Denhardt's solution, 10% dextran sulphate, 1 µg/ml carrier transfer RNA, and 10 mmol/l dithiothreitol. Post-hybridization washing was performed under stringent conditions that included ribonuclease A (25 µg/ml) treatment at 37°C for 30 min and a final stringency of 0.1x SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co) and exposed at 4°C until being developed after 2 weeks. The slides were stained with haematoxylin and eosin and were examined under the light microscope with bright- and dark-field illumination.
Follicle culture
Pre-ovulatory follicles (>800 µm in diameter) were isolated from ovaries collected at 48–52 h after PMSG injection, and follicle culture was performed as previously described (Lee et al., 1999). Follicles (15–20) were cultured in glass vials containing 800 µl Eagle's minimal essential medium (MEM) (Gibco, Grand Island, NY, USA) supplemented with penicillin, streptomycin, L-glutamine, and 0.1% BSA (wt/vol, Fraction V, Sigma Chemical Co) in the absence or presence of different hormones. Cultures were maintained in serum-free conditions for up to 24 h at 37°C under 5% CO2–95% O2. At the end of incubation, follicles were snap-frozen for RNA isolation.
Cloning of rat PACAP promoter
Cloning of rat PACAP promoter region was performed according to the manufacturer's instructions using GenomeWalker Kits (Clontech Laboratories Inc, Palo Alto, CA, USA). Briefly, the templates used for polymerase chain reaction (PCR) were five different genomic libraries of uncloned, adaptor-ligated DNA fragments provided in the kit. The primary PCR was performed with the template, genomic polymerase mix, the outer adaptor primer provided in the kit, and the gene-specific primer (5'-CGCTGGAATCACAACCAGAGAGGCATCAG-3'). The gene-specific primer was derived from the 5'-untranslated region of rat Pacap cDNA (Ogi et al., 1990) which corresponds to the exon 1B of the mouse Pacap gene (Yamamoto et al., 1998). The nested PCR was then performed using the diluted primary PCR reaction mixture as a template, the nested adaptor primer, and the nested gene-specific primer (5'-GGGGGACTTGTTTGCCGAAGCTAAAATTCC-3'). The PCR conditions for denaturation, annealing, and elongation for the first seven cycles were 94, 72, and 72°C for 25 s, 4 min and 4 min respectively, and for the subsequent 32 cycles were 94, 67, and 67°C for 25 s, 4 min, and 4 min respectively. Three overlapping PCR clones were identified and subcloned into the pGEM-T vector (Promega Corporation, Madison, WI, USA). The DNA sequence was determined by the dideoxy chain termination method using a sequencing kit (Amersham, Arlington Heights, IL, USA) and a DNA sequencer (Perkin-Elmer, Foster City, CA, USA).
Granulosa cell culture, transfection, and luciferase assays
Granulosa cells collected from pre-ovulatory follicles of PMSG-primed immature rat ovaries were cultured as described (Park-Sarge and Mayo, 1994) at a density of 0.5–1x106 cells per 2.5 ml of Dulbecco's modified Eagle's medium/F12 (DMEM/F12) medium supplemented with 10% fetal bovine serum (FBS; Gibco) in multiwell (60 mm) dishes in a humidified incubator at 37°C and 5% CO2. Granulosa cells were transiently transfected within 2 h after plating using a calcium phosphate precipitation method (Park-Sarge and Mayo, 1994) and 8 µg plasmid/well. To evaluate promoter activity, the 5'-flanking sequence (–1475 to –1 bp) of the rat PACAP promoter was ligated to pGL3 basic luciferase vector (pGL3-Luc; Promega Corporation) and named as PACAP-Luc. The plasmid DNA used for transfection contained PACAP-Luc, a fixed amount of pCMV-ß-galactosidase and a promoterless pGL3-Luc. After transfection for 15–20 h, the cells were washed and cultured in serum-free DMEM/F12 medium in the absence or presence of LH (200 ng/ml) with or without 10–5 mol/l RU486 or epostane for 7 h. The cells were then lysed in a buffer containing 1% Triton X-100, 25 mM HEPES, pH 7.8, 15 mmol/l MgSO4, 2 mmol/l EGTA, pH 8.0. Aliquots of the supernatant were analysed for ß-galactosidase and luciferase activities. For luciferase assays, the cell lysates (20 µl) were added to 100 µl of assay buffer (25 mmol/l HEPES, pH 7.8, 15 mmol/l MgSO4, 5 mmol/l ATP, 1 µg/ml BSA), and 20 µl of 1 mmol/l luciferin (Promega Corporation) was added using an automatic injector; emitted luminescence was measured using a LB9501 luminometer (Berthold, Germany) for 10 s. All data were corrected for the ß-galactosidase activity in each dish.
Data analysis
Statistical differences were assessed by one-way analysis of variance (ANOVA), followed by Student's t test; and P < 0.05 was considered to be statistically significant.
Results
Effect of RU486 or epostane on gonadotrophin-induced Pacap gene expression in PMSG/HCG-treated rat ovaries
To study the role of progesterone in gonadotrophin-induced Pacap gene expression, the progestin antagonist RU486 or an inhibitor of 3ß-hydroxysteroid dehydrogenase epostane was administered 1 h before HCG stimulation in PMSG-primed immature rats. Northern blot analysis of total ovarian RNA revealed the marked induction of Pacap mRNA 6 h after HCG stimulation (Figure 1A) as previously reported (Lee et al., 1999). Treatment with either RU486 (RU) or epostane (epo) suppressed the HCG-induced Pacap mRNA expression in a dose-dependent manner. Quantification of data, using the upper 3 kb PACAP transcript, showed 65% inhibition of HCG-stimulated Pacap mRNA values by RU486 or epostane at the maximal dose (Figure 1B) that effectively blocked ovulation. Quantification of the lower two transcripts (1.2 and 2.4 kb), showed the same levels of the inhibition by RU486 or epostane (data not shown).
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To determine the cell types expressing Pacap mRNA after RU486 treatment, antisense and sense cRNA probes for Pacap were generated for in-situ hybridization analysis. In ovaries of PMSG-primed immature rats followed by HCG stimulation for 6 h, high levels of Pacap mRNA were detected in all pre-ovulatory follicles (Figure 2A,B
) (Lee et al., 1999). However, in ovaries of PMSG/HCG-stimulated immature rats treated with RU486, 1 h before HCG, at a dose that effectively blocked ovulation (Tsafriri et al., 1987), the number of pre-ovulatory follicles expressing Pacap mRNA was greatly reduced (Figure 2C,D). Furthermore, treatment with RU486 resulted in the inhibition of Pacap mRNA expression in granulosa cells of pre-ovulatory follicles, but not in theca/interstitial cells (TI, arrowhead; Figure 2E). Ovarian sections hybridized with the sense PACAP riboprobe showed only background hybridization signals (Figure 2F).
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Effect of synthetic progestin on RU486- or epostane-suppressed Pacap gene expression in cultured pre-ovulatory follicles
To test whether the inhibitory effect of RU486 or epostane on LH-stimulated Pacap mRNA expression is reversed by the synthetic progestin R5020, pre-ovulatory follicles obtained from ovaries of rats primed for 2 days with PMSG were incubated in serum-free conditions in the presence of hormones. Increasing amounts of R5020 (0.5–50 µmol/l) were added to follicle cultures containing 200 ng/ml LH and 10–5 mol/l RU486 or epostane, and levels of Pacap mRNA expression were analysed after 7 h by Northern blot analysis. Treatment of pre-ovulatory follicles with either RU486 (Figure 3A
) or epostane (Figure 3B) suppressed the LH-stimulated Pacap mRNA expression by 60% (n = 2). Inclusion of R5020 caused a reversal of the inhibitory effect of RU486 (Figure 3A) or epostane (Figure 3B) on LH-induced Pacap mRNA expression. At 50 µmol/l R5020, the inhibitory effect of RU486 and epostane was completely reversed to the levels of LH-treated follicles. Treatment of follicles with R5020 alone had no effect on the expression of Pacap mRNA.
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Identification of a consensus progesterone response element (PRE) in the rat PACAP promoter
To examine further the molecular mechanisms by which progesterone regulates the LH-stimulated Pacap gene expression, genomic clones containing the 5'-portion of the rat Pacap gene were isolated by PCR using rat genomic libraries as a template. Three overlapping PCR clones were identified and sequenced. Figure 4
shows that the alignment of the 5'-portion of the rat and mouse Pacap genes (Yamamoto et al., 1998) revealed a high degree of sequence similarity (90%) including exon 1A, intron 1, exon 1B, and the 5'-flanking region. The 5'-flanking region of both rat and mouse Pacap genes contained several consensus elements reminiscent of some well-conserved consensus motifs involved in transcriptional control: two cAMP response elements (CRE), a TPA response element (TRE), and two growth hormone factor-1 (GHF-1) binding sites. In addition, a potential TATA box and PRE which shows 75% homology to the consensus sequence of PRE (Beato et al., 1989) was identified in the 5'-flanking region of the rat Pacap gene.
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Effect of RU486 or epostane on gonadotrophin- stimulated PACAP promoter activity in transfected granulosa cells
As a complementary approach and more direct way to assess the effect of progesterone on LH-stimulated Pacap gene expression, we analysed hormone-induced Pacap promoter activity in transfected granulosa cells. Granulosa cells obtained from pre-ovulatory follicles were transiently transfected with a fusion gene (PACAP-Luc), containing the 5'-flanking region of the rat Pacap gene ligated to the luciferase reporter gene, along with an internal control (pCMV-ß-gal) for transfection efficiency and a promoterless parental luciferase vector (pGL3-Luc). Treatment of the transfected granulosa cells with LH resulted in 7.7-fold increase in luciferase activity compared with that observed in cells transfected with PACAP-Luc and incubated in the absence of any hormones (Figure 5
). Co-treatment with RU486 (RU) or epostane (epo) partially suppressed the stimulatory effect of LH on luciferase activity. Treatment of these cells with RU486 or epostane alone had no effect on luciferase activity. Luciferase activity was not affected by any of these hormonal treatments in cells transfected with negative control promoterless vector, pGL3-Luc (data not shown).
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Discussion
Although the induction of progesterone and PR by the LH surge has been suggested to play a role in activating specific genes and thus regulating ovulation, target genes for PR action in the ovary have not been identified. Our results indicate that progesterone, acting through PR, plays a role in LH/HCG-induced Pacap gene expression in granulosa cells of pre-ovulatory follicles in the rat ovary, indicating that Pacap may be one of the target genes for PR action in the ovary. We previously observed the transient induction of Pacap mRNA by LH/HCG in granulosa cells of pre-ovulatory follicles (Lee et al., 1999). This gonadotrophin-induced Pacap mRNA expression in pre-ovulatory follicles was abolished by an antiprogestin RU486 both in vivo and in vitro, indicating the regulation of gonadotrophin-induced Pacap gene expression by progesterone. RU486 has been shown to antagonize progestin action by binding to PR and causing a conformational change in PR (Gass et al., 1998). Likewise, an inhibitor of progesterone biosynthesis, epostane, also inhibited the gonadotrophin-induced Pacap gene expression, suggesting that progesterone, acting through PR, plays a role in regulating Pacap gene expression. Moreover, RU486 or epostane suppressed the LH-stimulated PACAP promoter activity in transfected granulosa cells, providing a more direct way to demonstrate the role of progesterone and PR in regulating Pacap gene expression.
Although the induction of PR by LH has been shown to play an important role in the process of ovulation, the mechanism by which PR exerts its action remains unclear. The present study provides a clue for one way in which PR controls ovulation; i.e. it may be through the regulation of ovarian Pacap gene expression. Despite earlier reports showing a negligible role for progesterone in ovulation (Bullock and Kappauf, 1972; Holmes et al., 1985), evidence has been accumulating to document that progesterone is critical for regulating ovulation (Rondell, 1974) and luteinization (Rothchild, 1981) in the rat. Inhibitors of progesterone biosynthesis, such as epostane (Tsafriri et al., 1987; Espey et al., 1990; Tanaka et al., 1992) and aminoglutethimide (Lipner and Greep, 1971), can block LH induction of ovulation in vivo. Moreover, the administration of the antiprogestin RU486 in vivo has been shown to block ovulation (Tsafriri et al., 1987; Uilenbroek et al., 1992). These data indicate that progesterone, acting through PR, may play a role in the regulation of ovulation. Recent evidence for the presence of PR in the ovary has added credence to the action of this steroid in regulating ovulation. PR mRNA is transiently induced in granulosa cells of pre-ovulatory follicles after the LH surge, but not in granulosa cells of pre-ovulatory follicles before the LH surge in the rat (Park and Mayo, 1991). The fact that PR is only present transiently after the LH surge suggests that any specific receptor-mediated effects of progesterone on granulosa cells would have to occur during this period. PR mRNA was observed in detectable amounts by 2 h, reached a peak at 4–5 h, and declined by 8 h in rat granulosa cells of pre-ovulatory follicles (Park and Mayo, 1991; Natraj and Richards, 1993). Similar pattern of transient expression after LH/HCG has been observed in Pacap gene expression in the rat ovary (Gräs et al., 1996; Lee et al., 1999). The induction of Pacap mRNA in granulosa cells of pre-ovulatory follicles after gonadotrophin stimulation, however, appears to be delayed for a few hours, compared with that of PR mRNA; it was detected by 3 h, reached a peak by 6–9 h, and declined by 12 h (Lee et al., 1999). In addition, the induction of both PR and Pacap mRNA requires the same gonadotrophin-activated a cAMP-protein kinase A pathway (Park-Sarge and Mayo, 1994; Lee et al., 1999), suggesting that progesterone, acting through PR, may regulate Pacap gene expression. Such a view is supported by the present finding that treatment of PMSG-primed immature rats with RU486 or epostane partially inhibited gonadotrophin-induced ovarian Pacap gene expression in granulosa cells of pre-ovulatory follicles. In addition, the synthetic progestin R5020 could reverse the inhibitory effect of RU486 or epostane on Pacap gene expression in cultured pre-ovulatory follicles, further supporting the role of progesterone, acting through PR, in regulating Pacap gene expression. The present observation that RU486 treatment, at a dose that effectively blocked ovulation (Tsafriri et al., 1987), resulted in a partial inhibition of Pacap gene expression by reducing the number of pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells, raises the possibility that only those pre-ovulatory follicles expressing Pacap mRNA in their granulosa cells after RU486 treatment may eventually ovulate. If such a possibility exists, it appears plausible that PR plays a role in the process of ovulation by regulating Pacap gene expression. Thus, one major question that remains to be answered is whether PACAP is critical for regulating ovulation. Our recent observation, demonstrating the transient expression of Pacap type I receptor mRNA in granulosa cells of pre-ovulatory follicles after LH/HCG (Park et al., 2000), supports the possible role of PACAP in the process of ovulation as an autocrine factor. Interestingly, PR gene knock-out mice exhibit the failure to ovulate even when exogenous gonadotrophins are administered (Lydon et al., 1995). It would be of interest to test the Pacap gene expression and whether administration of gonadotrophins together with PACAP would overcome the failure to ovulate in PR gene knock-out mice. Indeed, PACAP has been shown to play a role in luteinization (Gräs et al., 1999). Because the induction of PR by gonadotrophins also plays, at least in part, a functional role in the luteinization process (Natraj and Richards, 1993), PR may control luteinization by regulating Pacap gene expression.
The role of progesterone in the regulation of gonadotrophin-induced Pacap gene expression was more directly proved at the transcriptional level by transfection studies. Although further studies are needed to determine the transcriptional start site, enhanced promoter activity after LH treatment could be observed in transfection studies using the cloned PACAP promoter. The 7.7-fold increase in LH-stimulated PACAP promoter activity in transfected granulosa cells observed in the present study does not seem to fully account for the marked induction of endogenous Pacap mRNA observed in these same cells. One likely explanation for this difference is that the 1474 bp fragment of rat PACAP promoter region used for these studies may be not sufficient for maximal responsiveness to LH that activates the cAMP pathway. Studies on the analysis of the mouse PACAP promoter function using 3.3 kb fragment of the 5'-flanking region of the mouse Pacap gene have shown almost 17-fold increase in promoter activity by forskolin treatment (Yamamoto et al., 1998). The present observation, showing a partial inhibition by RU486 or epostane of the LH-stimulated PACAP promoter activity, is in agreement with the present data demonstrating a partial inhibition by RU486 or epostane of the LH/HCG-stimulated Pacap gene expression in pre-ovulatory follicles. This partial inhibition by RU486 or epostane of the LH-stimulated PACAP promoter activity suggests that LH may induce Pacap gene expression by activating other pathway(s) in addition to the PR. Indeed, blockade of phospholipase A2 pathway has also been shown to inhibit the LH induction of Pacap gene expression in pre-ovulatory follicles (our own observations). Alternatively, the present observation that RU486 blocked Pacap gene expression in some but not all follicles raises the possibility that PR activation may be only an indirect mediator leading to transcriptional activation of the PACAP promoter, and may need the interaction with specific co-factors. Indeed, recent studies suggest that the altered conformation in PR induced by antagonists impairs the ability of receptors to interact with co-activator (Smith et al., 1996). Overexpression of the PR protein in the transfected HeLa cells did not affect the promoter activities of the Pacap gene (our own observations), suggesting that different cell types contain different levels of specific co-activators or co-repressors necessary for PR action (Kraus et al., 1993, 1994). It has been suggested that the relative amounts of co-activators and co-repressors may contribute to the partial agonist or antagonist activity of a ligand (Jackson et al., 1997; Smith et al., 1997).
In summary, this study shows that progesterone, acting through PR, plays a role in gonadotrophin-stimulated Pacap gene expression in granulosa cells of rat pre-ovulatory follicles. Whether PR plays a role in the process of ovulation by regulating Pacap gene expression remains to be clarified.
Acknowledgments
The authors would like to thank Dr Aaron J.W.Hsueh (Stanford University School of Medicine, Stanford, CA, USA) for providing R5020. This work was supported by KOSEF grants 97–04–01–06–01–3 and HRC-98k1-0405, Republic of Korea
Notes
4 To whom correspondence should be addressed 
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Submitted on September 20, 1999; accepted on December 21, 1999.
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