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Endocrinology Vol. 140, No. 11 5185-5194
Copyright © 1999 by The Endocrine Society

ARTICLES

Role of Progesterone Receptor Activation in Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in Rat Ovary1

Chemyong Ko, Yong-Ho In2 and Ok-Kyong Park-Sarge3

Department of Physiology, University of Kentucky, Lexington, Kentucky 40536

Address all correspondence and requests for reprints to: Dr. Ok-Kyong Park-Sarge, Department of Physiology, University of Kentucky, Lexington, Kentucky 40536-0084. E-mail: okps{at}pop.uky.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semiquantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT is well known that the ovarian steroid progesterone profoundly affects the physiology of female reproductive tissues (1, 2). At the ovarian level, progesterone modulates follicular activity (3, 4), ovulation and luteinization (5, 6), and luteal functions (7, 8). The intracellular progesterone receptor (PR), which belongs to the ligand-induced, hormonally regulated nuclear transcription factor family, is known to mediate many, if not all, progesterone actions (9), although there are other progesterone-binding proteins that have been implicated in mediating progesterone actions (10, 11). PR messenger RNA (mRNA) and protein have been localized to various ovarian cell types depending on species (12, 13, 14, 15, 16, 17); however, one consistent cellular localization for PR mRNA and protein is luteinizing granulosa cells. These cells transiently express the PR gene in response to the preovulatory gonadotropin surge as experimentally shown in rat (12), pig (13), and rhesus monkey (14). These results led us (12) and others (13, 14) to propose the hypothesis that PR expression in luteinizing granulosa cells plays a critical role in successful ovulation. Consistent with this hypothesis is the phenotype of PR -/- null mutant mice (18, 19). These mice can produce preovulatory follicles in response to an exogenous bolus of PMSG. However, these animals do not ovulate even after a bolus of exogenous human CG (hCG) in a well established superovulation protocol, indicating that ovulation does not occur in the absence of functional PRs. In light of this functional importance of PRs for ovulation, we wanted to dissect the molecular mechanisms by which progesterone regulates ovulatory processes. Because PRs are transcription factors, we reasoned that PR activation should regulate a cascade of gene expression leading to follicular rupture. In this study, we report the identification of the pituitary adenylate cyclase activating polypeptide (PACAP) as a progesterone-induced gene in granulosa cells. We further examined the temporal and spatial correlation between PR and PACAP mRNA in granulosa cells, ovaries of immature rats treated with exogenous gonadotropins, and ovaries of adult rats undergoing the endogenous gonadotropin surge during 4-day estrous cycles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Unless specifically stated, all molecular biological enzymes were obtained from New England Biolabs, Inc. (Beverly, MA). DMEM, Hams-F12, and antibiotics for tissue culture were from Life Technologies, Inc. (Gaithersburg, MD), and all general reagents were from Sigma Chemical Co. (St. Louis, MO). All radioisotopes were from New England Nuclear (Boston, MA). Oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

Animals and hormone treatments
In this study, two sets of animals were used: immature female rats treated with gonadotropins and sexually mature adult female rats exhibiting regular 4-day estrous cycles. All animals were handled according to the NIH guidelines for care and use of animals.

Immature female rats. Twenty-one-day-old Sprague Dawley female pups with nursing mothers were purchased from Harlan Breeding Company (Indianapolis, IN) and housed in a photoperiod of 14-h light/10-h darkness with lights on at 0500 h. At 22 or 23 days of age, rats were injected sc with 10 IU PMSG (Sigma Chemical Co.) in 0.1 ml PBS. Forty-eight hours later, rats were injected with 10 IU of hCG (Sigma Chemical Co.) in 0.1 ml PBS. Rats were killed by decapitation at various time points throughout the hormone treatment, and their ovaries were isolated, frozen on dry ice, and stored until use at -80 C.

Adult female rats. Adult female Sprague Dawley rat (150–180 g body weight) were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and housed as above. Estrous cyclic stages were determined by daily examinations of vaginal cytology, and only animals demonstrating at least two consecutive 4-day cycles were used for the experiments. On proestrus, rats were killed by decapitation at 1400 h, 1600 h, 1800 h, 2000 h, and 2200 h. Ovaries were collected for mRNA measurements by in situ hybridization and RT-PCR. Trunk blood was collected for determination of serum LH concentration by RIA. The LH surge peaked at 1800 h among these animals.

Granulosa cell isolation and culture
Granulosa cells of PMSG (10 IU, 48 h)-primed immature rats were isolated by the method of follicular puncture, with minor modifications (20, 21). Ovaries were collected in cold serum-free 4F medium consisting of 15 mM HEPES (pH 7.4), 50% DMEM, and 50% Ham’s F12 with bovine transferrin (5 µg/ml), human insulin (2 mg/ml), hydrocortisone (40 ng/ml), and antibiotics. Surrounding fat was removed using two sharp forceps, and the ovaries were incubated in warm (37 C) 4F medium containing 0.5 M sucrose and 10 mM EGTA for 20–30 min. Ovaries were washed three times in fresh 4F medium, and individual follicles were punctured using 23-gauge needles under a Leica Corp. (Deerfield, IL) dissection microscope. Cells were collected, counted using trypan blue, and plated in 4F medium supplemented with 5% FBS (Life Technologies, Inc.) at a density of approximately 1 x 106 cells per 60-mm dish, and incubated in the humidified atmosphere of 5% CO2 at 37 C. Five hours later, cells were treated with various hormones and reagents. Unless specifically stated, cells were harvested 6 h after hormone treatments and collected in a guanidium thiocyanate solution for RNA isolation (22).

RT-PCR
Total RNA from whole ovaries and granulosa cell cultures was purified by homogenization in a guanidium thiocyanate solution and centrifugation through a cesium-chloride gradient ultracentrifuge. RT-PCR was performed essentially as previously described (12, 21). Each PCR was carefully monitored to ensure that PCR products reflected the amount of input RNA in a linear range. Total RNA (1–2 µg) was reverse-transcribed at 37 C in a 20-µl volume using random hexamer (500 ng) and mouse mammary leukemia virus (MMLV) reverse transcriptase (10 U) (New England Biolabs, Inc.). Complementary DNA (cDNA) samples (2 µl) were used for subsequent PCR amplification of PR, PACAP, and S16 cDNAs using oligonucleotide primer pairs based upon the published rat PR (21) (5-CAAGACTGCCCCTCCCGACCA-3' and 5'-GGCTGCTGAGATGGCTTCAC-3', 810 bp), rat PACAP gene (23) (5'-GTGAAGATG CCGTCCGAGTGG-3' and 5'-CTTTGCCCGCCGTCCTATTTA-3', 452 bp), and rat S16 (24) (5'-TCCAAGGGTCCGCTGCAGTC-3' and 5'-TCCAAG GGTCCGCTGCAGTC-3', 100 bp). A 25-µl mix containing the primers (200 ng each), {alpha}-32P-dCTP (2 µCi at 3,000 Ci/mmol), and Taq DNA polymerase (2.5 U) in 1x PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin) were added to each cDNA sample and overlaid with light mineral oil. Amplification was carried out for 20 cycles using an annealing temperature of 60 C on a Perkin-Elmer thermocycler (Perkin-Elmer Corp., Norwalk, CT). The samples were then electrophoresed on an 8% nondenaturing polyacrylamide gel. The intensity of each band was analyzed using a PhosphorImager and ImageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale, CA). PR and PACAP signals were normalized to that of the ribosomal protein S16 internal control.

In situ hybridization
Frozen ovaries were cut in 20-µm sections using a MICROM HM 505 E cryostat (Microm Labogerate GmbH, Waldorf, Germany) and mounted onto Superfrost/Plus Microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were fixed, pretreated, and hybridized with antisense and sense RNA probes as previously described (12, 21). [35S]UTP-labeled RNA probes were synthesized from the rat PR-1 and the clone pSP72-OKPS#9 (rat PACAP, see Results) using T7 or SP6 polymerase. RNA probes [2 x 107 cpm/ml in hybridization buffer: 50% formamide, 5x SSPE, 2x Denhardt’s reagent, 10% dextran sulfate, 0.1% SDS, and 100 µg/ml yeast transfer RNA (tRNA)] were applied to sections and the sections were incubated in a humidity chamber at 47 C for 16–18 h. After hybridization, sections were treated with ribonuclease A (RNase A, 20 µg/ml) at 37 C for 30 min, washed in increasingly lower concentrations of SSC down to 0.1x SSC at 55 C, and dehydrated through an ethanol series. Slides were then exposed to Kodak XAR-5 film for 2 days and processed for liquid emulsion autoradiography using NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) for 2 weeks. Developed sections were stained using hematoxylin and photographed using an Axioskop microscope (Carl Zeiss, Jena, Germany).

PCR-based subtraction cloning and sequencing
To identify PR-induced genes in granulosa cells, we isolated poly A+ RNA of differentiated granulosa cells that were prepared from PMSG-treated (10 IU, sc, 40 h) immature rats and cultured for 6 h in the presence of forskolin (10-5 M) plus PR ligand [progesterone (10-7 M) or ZK-98288 (10-6 M)]. Cells were lysed in a guanidium thiocynate solution, and total RNA was isolated through a cesium chloride gradient ultracentrifugation. Poly A+ RNA was selected using an Oligotexôm RNA Purification System (QIAGEN, Chatsworth, CA) and used for a subtraction cloning using a PCR-Select cDNA Subtraction kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the manufacturer’s protocol. The first-strand cDNA was made using cDNA-synthesis primer (5'-TTTTGTACAAGCTT-3') and MMLV reverse transcriptase. Upon synthesis of the second strand cDNA, double-stranded cDNA was digested with RsaI. RsaI-digested cDNA blunt ends were ligated to adaptors I (5'-CTTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3') or II (5'-CTTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCC-GAGGT-3') to serve as the tester cDNA. First and second hybridization was performed using unligated RsaI-digested cDNA blunt ends (driver cDNA) in excess. This strategy should result in combinations of hybrids between tester and driver cDNAs. Use of primers for selecting only those hybrids enriched in differentially expressed genes is the basis of subtraction. Others (25, 26, 27) have successfully used similar approaches to clone differentially expressed genes. The subtracted and PCR-amplified cDNA inserts were ligated to the T-overhanged arms of the vector PCR2.1 (CLONTECH Laboratories, Inc.). This approach resulted in approximately 300 potentially subtracted clones, including the clone OKPS 9. DNA sequences of the clone OKPS 9 were determined with a Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham Pharmacia Biotech, Arlington Heights, IL) using M13 forward and backward primers.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PACAP mRNA expression requires PR activation in cultured granulosa cells
To identify PR downstream genes, we adopted a PCR-based subtraction cloning approach (CLONTECH Laboratories, Inc.) using poly A+ RNA of differentiated granulosa cells that were cultured for 6 h in the presence of forskolin (10-5 M) plus PR ligand progesterone (10-7 M, tester) or ZK-98299 (10-6 M, driver). Forskolin was used to induce PR mRNA (21, 28) and protein (29) as demonstrated previously. Progesterone-occupied PRs should stimulate or inhibit transcription of downstream target genes. Because ZK-98299 has been reported to disrupt normal interaction between PR and target DNA and thus gene regulation (30), we reasoned that ZK98299 would effectively prevent transcriptional modulation of progesterone-target genes in granulosa cells. Thus, cDNAs enriched in progesterone-treated cells should represent progesterone-stimulated genes in granulosa cells. Initial subtraction resulted in approximately 300 clones, some of which were analyzed by DNA sequencing. One such clone was the clone number 9 (OKPS 9) with a 517-bp cDNA insert that included the flanking nested PCR primer sequences (5'-TCGAGCGGCCGCCCGGGCAGGT-3' and 5'-AGCGTGGTCGCGGCCGAGGT-3'). Blast search using the 475-bp sequence identified the PACAP mRNA of several species including rats and mice with high homology. Figure 1Go shows alignment of DNA sequences among the clone OKPS 9, rat PACAP mRNA (GenBank accession number M63006; Ref. 23), mouse PACAP mRNA (GenBank accession number D14716; Ref. 31), and human PACAP mRNA (GenBank accession number X60435; Ref. 32). The N terminus (369 bp) of the clone OKPS 9 is identical to the corresponding region of the rat PACAP (2114–2483 bp). Interestingly, the C terminus (180 bp) of this clone is different from the corresponding region of the rat PACAP (2502–2681 bp), although it shows a clear homology to the corresponding region of the mouse (31) and human (32) PACAP mRNA sequences. The nucleotide sequence of this unmatched region of the previously published rat PACAP mRNA (23) shows a high homology (93%) with the cosmid L174G8 (EMBL accession number Z69638) in a reverse orientation. Taken together, these results indicate that the clone OKPS 9 is the PACAP mRNA or its derivative and that the PACAP is enriched in luteinizing granulosa cells upon PR activation.



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  		Figure 1. The subtracted clone OKPS 9 encodes the PACAP. The clone OKPS 9 was isolated using the PCR-based subtraction cloning approach using progesterone-treated granulosa cells as the tester sample and ZK-98299-treated granulosa cells as the driver sample (for details, see Materials and Methods). Blast search against the GenBank database identified the PACAP mRNA with high homology. Nucleotide sequences of the clone OKPS 9 are aligned and compared with corresponding regions of the previously reported PACAP mRNA sequences using ClustalW 1.7 software (Baylor College of Medicine, Human Genome Sequencing Center, Houston, TX). Numbers in the left side of the sequences indicate the position of the nucleotides in their reported sequences. The shading and boxing was performed using BOXSHADE 3.21 software (ISREC Informatics, Lausanne, Switzerland). The residues identical to the column consensus are indicated as inverse letters. Whereas the residues that are not identical but at least similar to the column consensus are indicated with gray backgrounds, the residues neither identical nor similar to the consensus are indicated in normal rendition. If there was no matching nucleotide, the positions are indicated by a dash (-).

 
To determine whether PACAP gene expression is controlled by PR activation, we performed a series of experiments using granulosa cells cultured in vitro. We initially examined whether hCG- or forskolin-induced cAMP stimulates PACAP mRNA in cultured granulosa cells. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats, treated with different doses of forskolin (10-6, 10-5, and 10-4 M) or hCG (0.5, 1, and 2 IU/ml), and analyzed for PACAP mRNA using semiquantitative RT-PCR assays. Results showed the PACAP mRNA was induced in a dose-dependent manner by both forskolin and hCG (Fig. 2Go), extending previous reports demonstrating the stimulatory effect of LH on PACAP mRNA in preovulatory follicles (33, 34). Forskolin as well as hCG induced PR mRNA in these cells, as shown previously for mRNA (21, 28). Interestingly, PR mRNA expression preceded PACAP mRNA expression (Fig. 3Go). PR mRNA levels reached the nadir within 3 h after forskolin treatment whereas PACAP mRNA levels reached its peak only after 6 h of treatment. Taken together, these results demonstrate that these two genes are expressed in luteinizing granulosa cells with a conceivable time gap, suggesting the possibility that cAMP-induced PR synthesis may be required for PACAP gene expression.



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  		Figure 2. hCG and forskolin increase PACAP mRNA levels in granulosa cells cultured in vitro. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and cultured for 6 h in the presence of vehicle (control), forskolin (FSK, panel A) or hCG (panel B) at various concentrations. Total RNA was isolated and analyzed for PACAP mRNA by semiquantitative RT-PCR assays using 20 cycles of amplification; ribosomal protein S16 mRNA was used as an internal control. Autoradiograms of polyacrylamide gels are shown on the upper part of each panel, and quantitated PACAP mRNA levels are displayed on the lower part of each panel. Band intensity was measured on a phosphoimager, and the PACAP signal was normalized to the S16 internal control for each sample. Values shown are the range of the two independent experiments along with the mean, being indicated by the bars. Hormone treatments are shown at the bottom.

 


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  		Figure 3. PR mRNA expression precedes PACAP mRNA expression in forskolin-treated granulosa cells. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and cultured for 1, 3, 6, 9, and 12 h in the presence of vehicle (control) or forskolin (FSK, 10 µM). Total RNA was isolated and analyzed for PR and PACAP mRNAs by semiquantitative RT-PCR assays using 20 cycles of amplification; ribosomal protein S16 mRNA was used as an internal control. Autoradiograms of polyacryamide gels are shown on the upper part of each panel, and quantitated PACAP mRNA levels are displayed on the lower part of each panel. Band intensity was measured on a phosphoimager, and the PACAP signal was normalized to the S16 internal control for each sample. Values shown are the range of two independent experiments along with the mean, indicated by the bars.

 
cAMP-induced PR mRNA expression does not require ongoing synthesis of new proteins as demonstrated previously (21) and, thus, we reasoned that if cAMP-induced PR synthesis is prerequisite for PACAP gene expression, cycloheximide, a protein synthesis inhibitor, should block cAMP-induced PACAP mRNA expression in granulosa cells. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and were pretreated with forskolin (10 µM) in the absence or presence of cycloheximide (1 or 10 µg/ml) for 6 h, and RNA was analyzed for PACAP mRNA expression using RT-PCR assays (Fig. 4Go). To ensure the effectiveness of cycloheximide, cells were pretreated with cycloheximide 1 h before hormone treatment. Cycloheximide effectively blocked protein synthesis to less than 2% of the level of the vehicle-treated control, as determined by [35S]Met incorporation analysis (100.00 ± 7.00% for control, 1.62 ± 0.06% for 10 µg/ml cycloheximide, n = 3). Under this condition, cycloheximide treatment suppressed forskolin-induced PACAP mRNA levels in a dose-dependent manner. This inhibitory effect of cycloheximide on forskolin-induced PACAP mRNA levels is unlikely due to cytotoxic effect of cycloheximide because the internal control S16 mRNA expression was not affected by the cycloheximide treatment. In addition, PR mRNA was not affected either (Ref. 21 and data not shown). To further determine the involvement of functional PR in cAMP-induced PACAP mRNA expression in granulosa cells, we examined the effect of ZK98299, a PR antagonist. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and treated with ZK98299 (1, 10, or 100 µM) or vehicle for 1 h. The cells were then treated with forskolin (10 µM) or hCG (1 IU/ml). After the 6-h hormone treatment, RNA was isolated for PACAP mRNA levels by RT-PCR assays. In these cells, ZK98299 (1–100 µM) effectively blocked forskolin-induced PACAP mRNA expression in a dose-dependent manner (Fig. 5AGo). In addition, ZK98299 suppressed hCG-induced PACAP mRNA expression (Fig. 5BGo), demonstrating that PR activation is critical for LH-induced PACAP gene expression in granulosa cells.



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  		Figure 4. Forskolin-induced granulosa cell PACAP mRNA expression requires new protein synthesis. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and cultured for 6 h with vehicle (control) or forskolin (FSK, 10 µM) in the presence or absence of cycloheximide, a protein synthesis inhibitor (1 or 10 µg/ml, Cyclo). For those cultures treated with cycloheximide, cells were pretreated with different doses of cycloheximide for 1 h before incubation for 6 h. Total RNA was isolated and analyzed for PR and PACAP mRNA by semiquantitative RT-PCR assays using 20 cycles of amplification; ribosomal protein S16 mRNA was used as an internal control. Autoradiograms of polyacryamide gels are shown on the upper part of each panel, and quantitated PACAP mRNA levels are displayed on the lower part of each panel. Band intensity was measured on a phosphoimager, and the PACAP signal was normalized to the S16 internal control for each sample. Values shown are the range of the two independent experiments along with the mean, indicated by the bars. Hormone treatments are shown at the bottom.

 


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  		Figure 5. ZK98299, a PR antagonist, blocks forskolin- or hCG-induced PACAP mRNA expression. Granulosa cells were isolated from PMSG (10 IU, 48 h)-primed immature rats and cultured for 6 h in the presence of vehicle (control) or forskolin (FSK, 10 µM) in the presence of ZK98299 at various concentrations (A). Similarly, cells were cultured for 6 h in the presence of vehicle (-), forskolin (FSK, 10 µM), or hCG (10 IU/ml) along with ZK98299 (10 µM). Total RNA was isolated and analyzed for PR and PACAP mRNA by semiquantitative RT-PCR assays using 20 cycles of amplification; ribosomal protein S16 mRNA was used as an internal control. Autoradiograms of polyacryamide gels are shown on the upper part of each panel, and quantitated PACAP mRNA levels are displayed on the lower part of each panel. Band intensity were measured on a phosphoimager, and the PACAP signal was normalized to the S16 internal control for each sample. Values shown are the range of two independent experiments along with the mean, indicated by bars. Hormone treatments are shown at the bottom.

 
PR and PACAP gene expression is temporally correlated in granulosa cells of preovulatory follicles in vivo
If functional PR activation is critical for PACAP gene expression in luteinizing granulosa cells, we reasoned that PR should be synthesized before PACAP in the granulosa cells of preovulatory follicles in vivo. Thus, we examined PR and PACAP mRNA expression in ovaries of 1) immature female rats treated with exogenous gonadotropins (Fig. 6Go); and 2) adult female rats undergoing the endogenous gonadotropin surges during regular 4-day estrous cycles (Fig. 7Go). We assessed the changes in PR and PACAP mRNA levels by performing semiquantitative RT-PCR assays in one ovary and in situ hybridization in the other ovary of the same rats. Figure 6Go, A and B, shows RT-PCR and in situ hybridization results on immature rats treated with gonadotropins, respectively. As shown previously (12, 21), PR mRNA was detected in granulosa cells of preovulatory follicles only after hCG treatment. Interestingly, PR mRNA expression reached maximal levels at 3 h of hCG treatment whereas PACAP mRNA was still undetectable. Ovarian PR mRNA expression decreased sharply in animals that received hCG for a longer duration (6–9 h), whereas PACAP mRNA gradually increased to its maximum during this period. This temporal gap in PR and PACAP mRNA levels was also seen at a cellular level as determined by in situ hybridization. Predominant expression of PR and PACAP mRNAs was observed in the granulosa cells of preovulatory follicles, in good agreement with previously reported results (12, 21, 33). A clear time gap between PR and PACAP gene expression was seen at the level of granulosa cells of preovulatory follicles. A better representation of this relationship at a higher magnification is given in Fig. 6CGo. Note that the same follicles show PR mRNA at a high level and PACAP mRNA at a low level in animals treated with PMSG (48 h) followed by hCG (3 h). In contrast, the same follicles show PR mRNA at a low level and PACAP mRNA at a high level in animals treated with PMSG (48 h) followed by hCG (6 h).



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  		Figure 6. PR mRNA expression precedes PACAP mRNA expression in vivo in PMSG-primed rat ovary. Immature rats at 22–23 days of age were primed with a single injection of PMSG (10 IU, 48 h) followed by a single injection of hCG (10 IU). Ovaries were collected from rats that were untreated (control), treated with PMSG for 24 h or 48 h, and treated with PMSG (48 h) followed by hCG (3, 6, 9, 12, and 24 h). One set of ovaries was used for RNA isolation and subsequent RT-PCR assays for PR and PACAP mRNA using 20 cycles of amplification (A); ribosomal protein S16 mRNA was used as an internal control. Values shown are the range of the two groups of animals along with the mean, which is indicated by bars. The other set of ovaries was used for in situ hybridization to localize PR and PACAP mRNAs on adjacent ovarian sections using 35S-labeled antisense RNA probes synthesized from the rPR-1 (15 ) or the clone OKPS 9. After hybridization, tissue sections were exposed to Kodak XAR-5 film (Eastman Kodak Co.) for 2 days, and the film was directly scanned for inversed images using the a Nikon LS-1000 film scanner (Nikon, Melville, NY) (B). After 2 weeks of exposure on NTB-2 liquid emulsion, sections were developed and photographed using dark-condenser at a 100x magnification (C).

 


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  		Figure 7. PR mRNA expression precedes PACAP mRNA expression in vivo in adult rat ovary during the preovulatory period. Adult female rats exhibiting at least two consecutive 4-day estrous cycles were killed by decapitation. Trunk blood was collected for serum LH concentrations as determined by LH RIA (the peak of the surge was seen at 1800 h). One set of ovaries was used for RNA isolation and subsequent RT-PCR assays for PR and PACAP mRNA using 20 cycles of amplification (A); ribosomal protein S16 mRNA was used as an internal control. Values shown are the range of two groups of animals along with the mean, indicated by bars. The other set of ovaries was used for in situ hybridization to localize PR and PACAP mRNAs on adjacent ovarian sections using 35S-labeled antisense RNA probes synthesized from the rPR-1 (15 ) or the clone OKPS 9. After hybridization, tissue sections were exposed to Kodak XAR-5 film (Eastman Kodak Co.) for 2 days, and the film was directly scanned for inverse images using the Nikon LS-1000 film scanner (B).

 
Figure 7Go, A and B, shows RT-PCR and in situ hybridization results on adult female rats undergoing the endogenous preovulatory LH surge during regular 4-day estrous cycles. We used only those rats exhibiting at least two consecutive 4-day cycles that showed the peak of the preovulatory LH surge at 1800 h as determined by LH RIA. As shown previously (12), PR mRNA expression is temporally correlated to the peak of the preovulatory LH surge. PR mRNA was detected predominantly in granulosa cells of preovulatory follicles during the narrow time window after the onset of the LH surge (1600–2000 h). PR mRNA levels peaked coincidentally with the peak of the LH surge (1800 h) but decreased sharply at 2000–2200 h. PACAP mRNA was barely detectable at 1600 h, increased at 1800 h, remained elevated at 2000 h, and was still detectable at 2200 h. This temporal gap in PR and PACAP mRNA levels in adult rats was also seen at a cellular level as determined by in situ hybridization. As reported previously (12), PR mRNA is localized to granulosa cells of preovulatory follicles during 1600–2000 h of proestrus. The peak expression was seen at 1800 h, in agreement with the RT-PCR results. PACAP mRNA localization by in situ hybridization revealed granulosa cells of preovulatory follicles as the primary site of PACAP gene expression. PACAP mRNA was barely detectable at 1600 h, peaked at 1800–2000 h, and remained detectable at 2200 h, whereas PR mRNA expression peaked at 1800 h and sharply declined at 2000 h. Taken together, these results show that, in rat ovary, both PR and PACAP mRNAs are localized to granulosa cells of preovulatory follicles but not to other cell types within the sensitivity of conventional in situ hybridization. These results further demonstrate that PR gene expression temporally correlated with the preovulatory LH surge precedes PACAP gene expression.

In summary, we showed that 1) the PACAP gene is a progesterone-induced gene in granulosa cells; 2) cAMP-induced PR synthesis precedes PACAP synthesis in granulosa cells; and 3) gonadotropin-induced PR synthesis precedes PACAP synthesis in granulosa cells of preovulatory follicles of gonadotropin-primed immature rats and adult proestrous rats.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The molecular and biochemical cascade underlying gonadotropin-induced ovulation appears to involve a variety of local regulators leading to the breakdown of the follicular basal membrane and the subsequent release of meiotically mature oocytes (35, 36, 37, 38, 39). One such local regulator is progesterone. The importance of this steroid in successful ovulation is supported by a long list of experimental evidence demonstrating the direct stimulatory effect of progesterone on the ovulation rate (5, 6). This intraovarian effect of progesterone is thought to be PR dependent because RU486, a progesterone antagonist, inhibits ovulation at the ovarian level and mimics a clinical luteinizing unruptured follicle syndrome in humans (40, 41, 42). Indeed, PRs are expressed in luteinizing granulosa cells of preovulatory follicles of many species examined thus far, during the narrow time window between the preovulatory gonadotropin surge and ovulation (12, 13, 14, 15, 16, 17, 29). Ablation of these PRs by gene knock-out technology resulted in animals with no gonadotropin-induced ovulation despite normal expression of gonadotropin receptors (18, 19, 43). Thus, the identification of PR downstream events should be essential for understanding the molecular mechanisms underlying successful ovulation and subsequent embryogenesis. We have isolated the PACAP cDNA in the subtraction cloning approach that was used to enrich progesterone-stimulated genes in luteinizing granulosa cells, providing one of the first steps toward understanding functional roles that PRs play in mammalian ovulation.

PACAP, originally thought to be expressed in pituitary cells and to play a crucial role in pituitary hormone synthesis (44, 45), is now known to be expressed and function in a variety of tissues (46). At the ovarian level in particular, PACAP mRNA and protein have been reported to be predominantly expressed in luteinizing granulosa cells of preovulatory follicles (33, 34) and, to a much lesser extent, by theca-interstitial cells of follicles at all stages (34). It has also been reported that luteal cells express PACAP proteins at all cyclic stages (34). Consistent with these previous results, we have also observed predominant expression of PACAP mRNA to luteinizing granulosa cells of preovulatory follicles. However, we have observed little PACAP mRNA expression in other cell types. This difference may be attributable to the sensitivity of different in situ hybridization approaches. Alternatively, it may be due to differences in cRNA probes corresponding to different regions of the PACAP mRNA. In fact, multiple transcripts, some of which are alternatively spliced (47), encode PACAP. The PACAP cDNA template used in this study is the subtracted clone OKPS 9. This clone contains the 3'-portion of the PACAP mRNA as expected from the cloning strategies. The 369 bp of this clone are identical to the corresponding region (2114–2483 bp) of the previously reported rat PACAP (GenBank accession number M63006; Ref. 23). However, the most 3'-end 180 bp sequences diverge from the corresponding region (2502–2681 bp) of this reported rat PACAP mRNA. It is possible that we have cloned PACAP transcripts with alternatively spliced exons. In this case, the clone OKPS 9 must represent the PACAP isoform corresponding to the previously reported mouse (31) and human (32) PACAP mRNA isoform.

Previous studies (33, 34) have demonstrated that gonadotropins and forskolin induce PACAP mRNA in luteinizing granulosa cells of preovulatory follicles in immature rats primed with gonadotropins. We have extended these studies by demonstrating the direct stimulatory effect of hCG and forskolin in cultured granulosa cells. We have also demonstrated the close temporal correlation between the preovulatory LH surge and PACAP mRNA in ovaries of adult rats during the preovulatory period, extending the previous result showing transient expression of ovarian PACAP mRNA during the early morning of estrus (34). In addition, our results demonstrate that PACAP gene expression occurs later than PR gene expression in luteinizing granulosa cells. Most importantly, our results demonstrate the inhibitory effect of ZK98299 on cAMP-induced PACAP expression. These results together identify PRs as one of the first sequential links between the preovulatory LH surge and PACAP gene expression. Because both LH and forskolin stimulate PR synthesis at the level of transcription in the absence of ongoing protein synthesis (Ref. 21 and this study), the inhibitory effect of cycloheximide on forskolin-induced PACAP mRNA expression in cultured granulosa cells must be due, at least in part, to the lack of PR synthesis. The PACAP promoter sequences available (48, 49) do not contain consensus PREs. Thus, the issue of whether PRs target the PACAP gene promoter directly or indirectly through other molecules remains to be determined. The results presented in this study do not exclude the possibility that more gene products may be involved between PRs and PACAP gene expression. One reported gene that requires PR activation in bovine granulosa cells is oxytocin (50). Thus, it will be important to elucidate whether oxytocin expression and PACAP expression are interdependent, leading to luteinization and/or ovulation.

Because the PR, a ligand-induced transcription factor, is indispensable for gonadotropin-induced ovulation and subsequent luteinization (18, 19, 43), the progesterone-stimulated gene PACAP must play a role in one or both of these events. Thus, the identification of PACAP as a progesterone-induced gene in luteinizing granulosa cells signifies an exciting window of new challenges. If PACAP should function as an autocrine and/or paracrine modulator, mediating progesterone action leading to ovulation and luteinization as we suggest here, PACAP needs to be processed and secreted to bind its own receptors in its target cells. Although the close link between PACAP synthesis and secretion has been demonstrated in cultured granulosa cells (51), the mechanisms underlying PACAP secretion are poorly understood. It is possible that PR activation also triggers cellular events leading to PACAP secretion. Alternatively, the preovulatory gonadotropin surge may independently trigger cellular events for protein secretion. Despite the lack of understanding what controls PACAP secretion, PACAP has been shown to affect several cellular events associated with ovulation. It stimulates intracellular cAMP accumulation and progesterone production in granulosa cells (51, 52), presumably through binding to PACAP receptors. The issue of whether both type I and type II receptors are present in granulosa cells of preovulatory follicles and mediate the effect of PACAP on progesterone synthesis has been addressed in previously published papers (53, 54) but still remains to be further examined. Nonetheless, it will be important to understand the relationship between PACAP and other local regulators such as proteases that have been implicated in ovulation (55, 56). Another aspect of ovulation that PACAP affects is meiotic maturation of oocytes (57, 58) presumably through its type I receptor (58). Because the PACAP type I receptor is expressed even in primordial germ cells (59), the LH-induced production of PACAP should dictate resumption of meiotic maturation of dictyate oocytes. Taken together, these results demonstrate that PR-induced PACAP synthesis may be a critical event for the initiation of gonadotropin-induced ovulation and oocyte meiotic maturation.


   Acknowledgments
 
We wish to thank Dr. Kyung-Soo Park for help in DNA sequencing and Ms. Lisa Savage for proofreading this manuscript. ZK98299 was kindly supplied by Dr. David Henderson at Schering AG, Germany.


   Footnotes
 
1 This work was supported by NIH Grants HD-30719 and HD-36879 (to O.K.P.S.). Back

2 Visiting graduate student scholar from the Department of Biochemistry and Molecular Biology, Hanyang University, South Korea. Back

3 Recipient of NIH Research Career Development Award HD-01135. Back

Received May 24, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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