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Role of Progesterone Receptor Activation in Pituitary Adenylate Cyclase

Activating Polypeptide Gene Expression in Rat Ovary1

Article  in  Endocrinology · December 1999

DOI: 10.1210/en.140.11.5185 · Source: PubMed

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Role of Progesterone Receptor Activation in PituitaryAdenylate Cyclase Activating Polypeptide GeneExpression in Rat Ovary*

CHEMYONG KO, YONG-HO IN†, AND OK-KYONG PARK-SARGE‡

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

ABSTRACTIt is well known that the pituitary gonadotropin surge induces

progesterone receptor (PR) gene expression in luteinizing granulosacells and that PR activation is critical for successful ovulation. Tofurther understand the molecular mechanism(s) by which PR plays arole critical for granulosa cell functions, we wanted to identify pro-gesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed dif-ferentially in granulosa cells that were challenged with forskolin inthe presence of progesterone or ZK98299. One such differentiallyexpressed clone was identified as the pituitary adenylate cyclaseactivating polypeptide (PACAP). To begin to understand the rela-tionship between PR activation and PACAP gene expression in lu-teinizing granulosa cells, we examined whether PR and PACAP mes-senger RNA (mRNA) expression is temporally correlated. In culturedgranulosa cells, both human CG and forskolin induced PR and PACAPmRNA levels in a dose-dependent manner, as determined by semi-quantitative RT-PCR assays. However, the peak expression for PRand PACAP mRNAs was observed at 3 h and 6 h after hormonetreatment, respectively. This time difference in cAMP-responsive ex-

pression of the PR and PACAP genes is due, at least in part, to therequirement of ongoing protein synthesis for PACAP expression, asdemonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PRsynthesis is prerequisite for PACAP expression, we examined theeffect of ZK98299, a specific PR antagonist, on cAMP-induced PACAPmRNA expression. This compound blocked cAMP-induced PACAPmRNA expression in a dose-dependent manner, indicating that PRactivation is required for PACAP gene expression in granulosa cells.We then compared cellular localization and hormonal regulation ofovarian PR and PACAP gene expression in immature rats treatedwith gonadotropins as well as in adult rats during the preovulatoryperiod by using in situ hybridization and semiquantitative RT-PCRassays. Results show that both PR and PACAP mRNAs are inducedin granulosa cells of preovulatory follicles by human CG, but that thePR gene is expressed before the PACAP gene. Taken together, theseresults demonstrate that PRs mediate the LH-induced PACAP geneexpression in rat granulosa cells. (Endocrinology 140: 5185–5194,1999)

IT is well known that the ovarian steroid progesteroneprofoundly affects the physiology of female reproductive

tissues (1, 2). At the ovarian level, progesterone modulatesfollicular activity (3, 4), ovulation and luteinization (5, 6), andluteal functions (7, 8). The intracellular progesterone receptor(PR), which belongs to the ligand-induced, hormonally reg-ulated nuclear transcription factor family, is known to me-diate many, if not all, progesterone actions (9), although thereare other progesterone-binding proteins that have been im-plicated in mediating progesterone actions (10, 11). PR mes-senger RNA (mRNA) and protein have been localized tovarious ovarian cell types depending on species (12–17);however, one consistent cellular localization for PR mRNAand protein is luteinizing granulosa cells. These cells tran-siently express the PR gene in response to the preovulatorygonadotropin surge as experimentally shown in rat (12), pig(13), and rhesus monkey (14). These results led us (12) andothers (13, 14) to propose the hypothesis that PR expression

in luteinizing granulosa cells plays a critical role in successfulovulation. Consistent with this hypothesis is the phenotypeof PR 2/2 null mutant mice (18, 19). These mice can producepreovulatory follicles in response to an exogenous bolus ofPMSG. However, these animals do not ovulate even after abolus of exogenous human CG (hCG) in a well establishedsuperovulation protocol, indicating that ovulation does notoccur in the absence of functional PRs. In light of this func-tional importance of PRs for ovulation, we wanted to dissectthe molecular mechanisms by which progesterone regulatesovulatory processes. Because PRs are transcription factors,we reasoned that PR activation should regulate a cascade ofgene expression leading to follicular rupture. In this study,we report the identification of the pituitary adenylate cyclaseactivating polypeptide (PACAP) as a progesterone-inducedgene in granulosa cells. We further examined the temporaland spatial correlation between PR and PACAP mRNA ingranulosa cells, ovaries of immature rats treated with exog-enous gonadotropins, and ovaries of adult rats undergoingthe endogenous gonadotropin surge during 4-day estrouscycles.

Materials and MethodsMaterials

Unless specifically stated, all molecular biological enzymes were ob-tained from New England Biolabs, Inc. (Beverly, MA). DMEM, Hams-F12, and antibiotics for tissue culture were from Life Technologies, Inc.

Received May 24, 1999.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@pop.uky.edu.

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

† Visiting graduate student scholar from the Department of Biochem-istry and Molecular Biology, Hanyang University, South Korea.

‡ Recipient of NIH Research Career Development Award HD-01135.

0013-7227/99/$03.00/0 Vol. 140, No. 11Endocrinology Printed in U.S.A.Copyright © 1999 by The Endocrine Society

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(Gaithersburg, MD), and all general reagents were from Sigma ChemicalCo. (St. Louis, MO). All radioisotopes were from New England Nuclear(Boston, MA). Oligonucleotides were synthesized by Integrated DNATechnologies, Inc. (Coralville, IA).

Animals and hormone treatments

In this study, two sets of animals were used: immature female ratstreated with gonadotropins and sexually mature adult female rats ex-hibiting regular 4-day estrous cycles. All animals were handled accord-ing to the NIH guidelines for care and use of animals.

Immature female rats. Twenty-one-day-old Sprague Dawley female pupswith nursing mothers were purchased from Harlan Breeding Company(Indianapolis, IN) and housed in a photoperiod of 14-h light/10-h dark-ness with lights on at 0500 h. At 22 or 23 days of age, rats were injectedsc with 10 IU PMSG (Sigma Chemical Co.) in 0.1 ml PBS. Forty-eighthours 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 pointsthroughout the hormone treatment, and their ovaries were isolated,frozen on dry ice, and stored until use at 280 C.

Adult female rats. Adult female Sprague Dawley rat (150–180 g bodyweight) were purchased from Charles River Laboratories, Inc. (Wil-mington, MA) and housed as above. Estrous cyclic stages were deter-mined by daily examinations of vaginal cytology, and only animalsdemonstrating at least two consecutive 4-day cycles were used for theexperiments. On proestrus, rats were killed by decapitation at 1400 h,1600 h, 1800 h, 2000 h, and 2200 h. Ovaries were collected for mRNAmeasurements by in situ hybridization and RT-PCR. Trunk blood wascollected for determination of serum LH concentration by RIA. The LHsurge peaked at 1800 h among these animals.

Granulosa cell isolation and culture

Granulosa cells of PMSG (10 IU, 48 h)-primed immature rats wereisolated by the method of follicular puncture, with minor modifications(20, 21). Ovaries were collected in cold serum-free 4F medium consistingof 15 mm HEPES (pH 7.4), 50% DMEM, and 50% Ham’s F12 with bovinetransferrin (5 mg/ml), human insulin (2 mg/ml), hydrocortisone (40ng/ml), and antibiotics. Surrounding fat was removed using two sharpforceps, and the ovaries were incubated in warm (37 C) 4F mediumcontaining 0.5 m sucrose and 10 mm EGTA for 20–30 min. Ovaries werewashed three times in fresh 4F medium, and individual follicles werepunctured 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 3 106 cells per 60-mm dish, andincubated in the humidified atmosphere of 5% CO2 at 37 C. Five hourslater, cells were treated with various hormones and reagents. Unlessspecifically stated, cells were harvested 6 h after hormone treatmentsand collected in a guanidium thiocyanate solution for RNA isolation(22).

RT-PCR

Total RNA from whole ovaries and granulosa cell cultures was pu-rified by homogenization in a guanidium thiocyanate solution and cen-trifugation through a cesium-chloride gradient ultracentrifuge. RT-PCRwas performed essentially as previously described (12, 21). Each PCRwas carefully monitored to ensure that PCR products reflected theamount of input RNA in a linear range. Total RNA (1–2 mg) was reverse-transcribed at 37 C in a 20-ml volume using random hexamer (500 ng)and mouse mammary leukemia virus (MMLV) reverse transcriptase (10U) (New England Biolabs, Inc.). Complementary DNA (cDNA) samples(2 ml) were used for subsequent PCR amplification of PR, PACAP, andS16 cDNAs using oligonucleotide primer pairs based upon the pub-lished rat PR (21) (5-CAAGACTGCCCCTCCCGACCA-39 and 59-GGCT-GCTGAGATGGCTTCAC-39, 810 bp), rat PACAP gene (23) (59-GTGAA-GATG CCGTCCGAGTGG-39 and 59-CTTTGCCCGCCGTCCTATTTA-39, 452 bp), and rat S16 (24) (59-TCCAAGGGTCCGCTGCAGTC-39 and59-TCCAAG GGTCCGCTGCAGTC-39, 100 bp). A 25-ml mix containingthe primers (200 ng each), a-32P-dCTP (2 mCi at 3,000 Ci/mmol), and TaqDNA polymerase (2.5 U) in 13 PCR buffer (10 mm Tris, pH 8.3, 50 mm

KCl, 1.5 mm MgCl2, 0.01% gelatin) were added to each cDNA sampleand overlaid with light mineral oil. Amplification was carried out for 20cycles using an annealing temperature of 60 C on a Perkin-Elmer ther-mocycler (Perkin-Elmer Corp., Norwalk, CT). The samples were thenelectrophoresed on an 8% nondenaturing polyacrylamide gel. The in-tensity of each band was analyzed using a PhosphorImager and Im-ageQuant version 3 software (Molecular Dynamics, Inc., Sunnyvale,CA). PR and PACAP signals were normalized to that of the ribosomalprotein S16 internal control.

In situ hybridization

Frozen ovaries were cut in 20-mm sections using a MICROM HM 505E cryostat (Microm Labogerate GmbH, Waldorf, Germany) andmounted onto Superfrost/Plus Microscope slides (Fisher Scientific,Pittsburgh, PA). Sections were fixed, pretreated, and hybridized withantisense and sense RNA probes as previously described (12, 21).[35S]UTP-labeled RNA probes were synthesized from the rat PR-1 andthe clone pSP72-OKPS#9 (rat PACAP, see Results) using T7 or SP6polymerase. RNA probes [2 3 107 cpm/ml in hybridization buffer: 50%formamide, 53 SSPE, 23 Denhardt’s reagent, 10% dextran sulfate, 0.1%SDS, and 100 mg/ml yeast transfer RNA (tRNA)] were applied to sec-tions and the sections were incubated in a humidity chamber at 47 C for16–18 h. After hybridization, sections were treated with ribonuclease A(RNase A, 20 mg/ml) at 37 C for 30 min, washed in increasingly lowerconcentrations of SSC down to 0.13 SSC at 55 C, and dehydratedthrough an ethanol series. Slides were then exposed to Kodak XAR-5film for 2 days and processed for liquid emulsion autoradiography usingNTB-2 emulsion (Eastman Kodak Co., Rochester, NY) for 2 weeks.Developed sections were stained using hematoxylin and photographedusing an Axioskop microscope (Carl Zeiss, Jena, Germany).

PCR-based subtraction cloning and sequencing

To identify PR-induced genes in granulosa cells, we isolated poly A1

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 presenceof forskolin (1025 m) plus PR ligand [progesterone (1027 m) or ZK-98288(1026 m)]. Cells were lysed in a guanidium thiocynate solution, and totalRNA was isolated through a cesium chloride gradient ultracentrifuga-tion. Poly A1 RNA was selected using an Oligotexom RNA PurificationSystem (QIAGEN, Chatsworth, CA) and used for a subtraction cloningusing 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 (59-TTTTGTA-CAAGCTT-39) and MMLV reverse transcriptase. Upon synthesis of thesecond strand cDNA, double-stranded cDNA was digested with RsaI.RsaI-digested cDNA blunt ends were ligated to adaptors I (59-CTTA-ATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-39)or II (59-CTTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCC-GAGGT-39) to serve as the tester cDNA. First and second hybridizationwas performed using unligated RsaI-digested cDNA blunt ends (drivercDNA) in excess. This strategy should result in combinations of hybridsbetween tester and driver cDNAs. Use of primers for selecting only thosehybrids enriched in differentially expressed genes is the basis of sub-traction. Others (25–27) have successfully used similar approaches toclone differentially expressed genes. The subtracted and PCR-amplifiedcDNA inserts were ligated to the T-overhanged arms of the vectorPCR2.1 (CLONTECH Laboratories, Inc.). This approach resulted in ap-proximately 300 potentially subtracted clones, including the clone OKPS9. DNA sequences of the clone OKPS 9 were determined with a ThermoSequenase radiolabeled terminator cycle sequencing kit (AmershamPharmacia Biotech, Arlington Heights, IL) using M13 forward and back-ward primers.

ResultsPACAP mRNA expression requires PR activation incultured granulosa cells

To identify PR downstream genes, we adopted a PCR-based subtraction cloning approach (CLONTECH Labora-

5186 PR AND PACAP IN OVARY Endo • 1999Vol 140 • No 11

tories, Inc.) using poly A1 RNA of differentiated granulosacells that were cultured for 6 h in the presence of forskolin(1025 m) plus PR ligand progesterone (1027 m, tester) orZK-98299 (1026 m, driver). Forskolin was used to induce PRmRNA (21, 28) and protein (29) as demonstrated previously.Progesterone-occupied PRs should stimulate or inhibit tran-scription of downstream target genes. Because ZK-98299 hasbeen reported to disrupt normal interaction between PR and

target DNA and thus gene regulation (30), we reasoned thatZK98299 would effectively prevent transcriptional modula-tion of progesterone-target genes in granulosa cells. Thus,cDNAs enriched in progesterone-treated cells should repre-sent progesterone-stimulated genes in granulosa cells. Initialsubtraction resulted in approximately 300 clones, some ofwhich were analyzed by DNA sequencing. One such clonewas the clone number 9 (OKPS 9) with a 517-bp cDNA insert

FIG. 1. The subtracted clone OKPS 9 encodes the PACAP. The clone OKPS 9 was isolated using the PCR-based subtraction cloning approachusing progesterone-treated granulosa cells as the tester sample and ZK-98299-treated granulosa cells as the driver sample (for details, seeMaterials and Methods). Blast search against the GenBank database identified the PACAP mRNA with high homology. Nucleotide sequencesof the clone OKPS 9 are aligned and compared with corresponding regions of the previously reported PACAP mRNA sequences using ClustalW1.7 software (Baylor College of Medicine, Human Genome Sequencing Center, Houston, TX). Numbers in the left side of the sequences indicatethe position of the nucleotides in their reported sequences. The shading and boxing was performed using BOXSHADE 3.21 software (ISRECInformatics, Lausanne, Switzerland). The residues identical to the column consensus are indicated as inverse letters. Whereas the residues thatare not identical but at least similar to the column consensus are indicated with gray backgrounds, the residues neither identical nor similarto the consensus are indicated in normal rendition. If there was no matching nucleotide, the positions are indicated by a dash (-).

PR AND PACAP IN OVARY 5187

that included the flanking nested PCR primer sequences(59-TCGAGCGGCCGCCCGGGCAGGT-39 and 59-AGCGT-GGTCGCGGCCGAGGT-39). Blast search using the 475-bpsequence identified the PACAP mRNA of several speciesincluding rats and mice with high homology. Figure 1 showsalignment of DNA sequences among the clone OKPS 9, ratPACAP mRNA (GenBank accession number M63006; Ref.23), mouse PACAP mRNA (GenBank accession numberD14716; Ref. 31), and human PACAP mRNA (GenBank ac-cession number X60435; Ref. 32). The N terminus (369 bp) ofthe clone OKPS 9 is identical to the corresponding region ofthe rat PACAP (2114–2483 bp). Interestingly, the C terminus(180 bp) of this clone is different from the correspondingregion of the rat PACAP (2502–2681 bp), although it showsa clear homology to the corresponding region of the mouse(31) and human (32) PACAP mRNA sequences. The nucle-otide sequence of this unmatched region of the previouslypublished rat PACAP mRNA (23) shows a high homology(93%) with the cosmid L174G8 (EMBL accession numberZ69638) in a reverse orientation. Taken together, these resultsindicate that the clone OKPS 9 is the PACAP mRNA or itsderivative and that the PACAP is enriched in luteinizinggranulosa cells upon PR activation.

To determine whether PACAP gene expression is con-trolled by PR activation, we performed a series of experi-ments using granulosa cells cultured in vitro. We initiallyexamined whether hCG- or forskolin-induced cAMP stim-ulates PACAP mRNA in cultured granulosa cells. Granulosacells were isolated from PMSG (10 IU, 48 h)-primed imma-ture rats, treated with different doses of forskolin (1026, 1025,and 1024 m) or hCG (0.5, 1, and 2 IU/ml), and analyzed forPACAP mRNA using semiquantitative RT-PCR assays. Re-sults showed the PACAP mRNA was induced in a dose-dependent manner by both forskolin and hCG (Fig. 2), ex-tending previous reports demonstrating the stimulatoryeffect 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. 3). PR mRNA levels reached the nadir within 3 h afterforskolin treatment whereas PACAP mRNA levels reachedits peak only after 6 h of treatment. Taken together, theseresults demonstrate that these two genes are expressed inluteinizing granulosa cells with a conceivable time gap, sug-gesting the possibility that cAMP-induced PR synthesis maybe required for PACAP gene expression.

cAMP-induced PR mRNA expression does not requireongoing synthesis of new proteins as demonstrated previ-ously (21) and, thus, we reasoned that if cAMP-induced PRsynthesis is prerequisite for PACAP gene expression, cyclo-heximide, a protein synthesis inhibitor, should block cAMP-induced PACAP mRNA expression in granulosa cells. Gran-ulosa cells were isolated from PMSG (10 IU, 48 h)-primedimmature rats and were pretreated with forskolin (10 mm) inthe absence or presence of cycloheximide (1 or 10 mg/ml) for6 h, and RNA was analyzed for PACAP mRNA expressionusing RT-PCR assays (Fig. 4). To ensure the effectiveness ofcycloheximide, cells were pretreated with cycloheximide 1 hbefore hormone treatment. Cycloheximide effectivelyblocked protein synthesis to less than 2% of the level of thevehicle-treated control, as determined by [35S]Met incorpo-ration analysis (100.00 6 7.00% for control, 1.62 6 0.06%for 10 mg/ml cycloheximide, n 5 3). Under this condition,cycloheximide treatment suppressed forskolin-inducedPACAP mRNA levels in a dose-dependent manner. Thisinhibitory effect of cycloheximide on forskolin-inducedPACAP mRNA levels is unlikely due to cytotoxic effect ofcycloheximide because the internal control S16 mRNA ex-pression was not affected by the cycloheximide treatment. Inaddition, PR mRNA was not affected either (Ref. 21 and datanot shown). To further determine the involvement of func-tional PR in cAMP-induced PACAP mRNA expression ingranulosa cells, we examined the effect of ZK98299, a PRantagonist. Granulosa cells were isolated from PMSG (10 IU,48 h)-primed immature rats and treated with ZK98299 (1, 10,

FIG. 2. hCG and forskolin increasePACAP mRNA levels in granulosa cellscultured in vitro. Granulosa cells wereisolated from PMSG (10 IU, 48 h)-primed immature rats and cultured for6 h in the presence of vehicle (control),forskolin (FSK, panel A) or hCG (panelB) at various concentrations. Total RNAwas isolated and analyzed for PACAPmRNA by semiquantitative RT-PCR as-says using 20 cycles of amplification; ri-bosomal protein S16 mRNA was used asan internal control. Autoradiograms ofpolyacrylamide gels are shown on theupper part of each panel, and quanti-tated PACAP mRNA levels are dis-played on the lower part of each panel.Band intensity was measured on a phos-phoimager, and the PACAP signal wasnormalized to the S16 internal controlfor each sample. Values shown are therange of the two independent experi-ments along with the mean, being indi-cated by the bars. Hormone treatmentsare shown at the bottom.

5188 PR AND PACAP IN OVARY Endo • 1999Vol 140 • No 11

or 100 mm) or vehicle for 1 h. The cells were then treated withforskolin (10 mm) or hCG (1 IU/ml). After the 6-h hormonetreatment, RNA was isolated for PACAP mRNA levels byRT-PCR assays. In these cells, ZK98299 (1–100 mm) effectivelyblocked forskolin-induced PACAP mRNA expression in adose-dependent manner (Fig. 5A). In addition, ZK98299 sup-pressed hCG-induced PACAP mRNA expression (Fig. 5B),demonstrating that PR activation is critical for LH-inducedPACAP gene expression in granulosa cells.

PR and PACAP gene expression is temporally correlated ingranulosa cells of preovulatory follicles in vivo

If functional PR activation is critical for PACAP gene ex-pression in luteinizing granulosa cells, we reasoned that PRshould be synthesized before PACAP in the granulosa cellsof preovulatory follicles in vivo. Thus, we examined PR andPACAP mRNA expression in ovaries of 1) immature femalerats treated with exogenous gonadotropins (Fig. 6); and 2)adult female rats undergoing the endogenous gonadotropinsurges during regular 4-day estrous cycles (Fig. 7). We as-sessed the changes in PR and PACAP mRNA levels by per-

forming semiquantitative RT-PCR assays in one ovary and insitu hybridization in the other ovary of the same rats. Figure6, A and B, shows RT-PCR and in situ hybridization resultson immature rats treated with gonadotropins, respectively.As shown previously (12, 21), PR mRNA was detected ingranulosa cells of preovulatory follicles only after hCG treat-ment. Interestingly, PR mRNA expression reached maximallevels at 3 h of hCG treatment whereas PACAP mRNA wasstill undetectable. Ovarian PR mRNA expression decreasedsharply in animals that received hCG for a longer duration(6–9 h), whereas PACAP mRNA gradually increased to itsmaximum during this period. This temporal gap in PR andPACAP mRNA levels was also seen at a cellular level asdetermined by in situ hybridization. Predominant expressionof PR and PACAP mRNAs was observed in the granulosacells of preovulatory follicles, in good agreement with pre-viously reported results (12, 21, 33). A clear time gap betweenPR and PACAP gene expression was seen at the level ofgranulosa cells of preovulatory follicles. A better represen-tation of this relationship at a higher magnification is givenin Fig. 6C. Note that the same follicles show PR mRNA at a

FIG. 3. PR mRNA expression precedes PACAP mRNA expression inforskolin-treated granulosa cells. Granulosa cells were isolated fromPMSG (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 mM).Total RNA was isolated and analyzed for PR and PACAP mRNAs bysemiquantitative RT-PCR assays using 20 cycles of amplification;ribosomal protein S16 mRNA was used as an internal control. Au-toradiograms of polyacryamide gels are shown on the upper part ofeach panel, and quantitated PACAP mRNA levels are displayed onthe lower part of each panel. Band intensity was measured on aphosphoimager, and the PACAP signal was normalized to the S16internal control for each sample. Values shown are the range of twoindependent experiments along with the mean, indicated by the bars.

FIG. 4. Forskolin-induced granulosa cell PACAP mRNA expressionrequires new protein synthesis. Granulosa cells were isolated fromPMSG (10 IU, 48 h)-primed immature rats and cultured for 6 h withvehicle (control) or forskolin (FSK, 10 mM) in the presence or absenceof cycloheximide, a protein synthesis inhibitor (1 or 10 mg/ml, Cyclo).For those cultures treated with cycloheximide, cells were pretreatedwith different doses of cycloheximide for 1 h before incubation for 6 h.Total RNA was isolated and analyzed for PR and PACAP mRNA bysemiquantitative RT-PCR assays using 20 cycles of amplification;ribosomal protein S16 mRNA was used as an internal control. Au-toradiograms of polyacryamide gels are shown on the upper part ofeach panel, and quantitated PACAP mRNA levels are displayed onthe lower part of each panel. Band intensity was measured on aphosphoimager, and the PACAP signal was normalized to the S16internal control for each sample. Values shown are the range of thetwo independent experiments along with the mean, indicated by thebars. Hormone treatments are shown at the bottom.

PR AND PACAP IN OVARY 5189

high level and PACAP mRNA at a low level in animalstreated with PMSG (48 h) followed by hCG (3 h). In contrast,the same follicles show PR mRNA at a low level and PACAPmRNA at a high level in animals treated with PMSG (48 h)followed by hCG (6 h).

Figure 7, A and B, shows RT-PCR and in situ hybridizationresults on adult female rats undergoing the endogenous pre-ovulatory LH surge during regular 4-day estrous cycles. Weused only those rats exhibiting at least two consecutive 4-daycycles that showed the peak of the preovulatory LH surge at1800 h as determined by LH RIA. As shown previously (12),PR mRNA expression is temporally correlated to the peak ofthe preovulatory LH surge. PR mRNA was detected pre-dominantly in granulosa cells of preovulatory follicles dur-ing the narrow time window after the onset of the LH surge(1600–2000 h). PR mRNA levels peaked coincidentally withthe peak of the LH surge (1800 h) but decreased sharply at2000–2200 h. PACAP mRNA was barely detectable at 1600 h,increased at 1800 h, remained elevated at 2000 h, and was stilldetectable at 2200 h. This temporal gap in PR and PACAPmRNA levels in adult rats was also seen at a cellular level asdetermined by in situ hybridization. As reported previously(12), PR mRNA is localized to granulosa cells of preovulatoryfollicles during 1600–2000 h of proestrus. The peak expres-sion was seen at 1800 h, in agreement with the RT-PCRresults. PACAP mRNA localization by in situ hybridization

revealed granulosa cells of preovulatory follicles as the pri-mary site of PACAP gene expression. PACAP mRNA wasbarely detectable at 1600 h, peaked at 1800–2000 h, andremained detectable at 2200 h, whereas PR mRNA expres-sion peaked at 1800 h and sharply declined at 2000 h. Takentogether, these results show that, in rat ovary, both PR andPACAP mRNAs are localized to granulosa cells of preovu-latory follicles but not to other cell types within the sensi-tivity of conventional in situ hybridization. These resultsfurther demonstrate that PR gene expression temporally cor-related with the preovulatory LH surge precedes PACAPgene expression.

In summary, we showed that 1) the PACAP gene is aprogesterone-induced gene in granulosa cells; 2) cAMP-in-duced PR synthesis precedes PACAP synthesis in granulosacells; and 3) gonadotropin-induced PR synthesis precedesPACAP synthesis in granulosa cells of preovulatory folliclesof gonadotropin-primed immature rats and adult proestrousrats.

Discussion

The molecular and biochemical cascade underlying gona-dotropin-induced ovulation appears to involve a variety oflocal regulators leading to the breakdown of the follicularbasal membrane and the subsequent release of meiotically

FIG. 5. ZK98299, a PR antagonist, blocks forskolin- or hCG-induced PACAP mRNA expression. Granulosa cells were isolated from PMSG (10IU, 48 h)-primed immature rats and cultured for 6 h in the presence of vehicle (control) or forskolin (FSK, 10 mM) in the presence of ZK98299at various concentrations (A). Similarly, cells were cultured for 6 h in the presence of vehicle (2), forskolin (FSK, 10 mM), or hCG (10 IU/ml)along with ZK98299 (10 mM). Total RNA was isolated and analyzed for PR and PACAP mRNA by semiquantitative RT-PCR assays using 20cycles of amplification; ribosomal protein S16 mRNA was used as an internal control. Autoradiograms of polyacryamide gels are shown on theupper part of each panel, and quantitated PACAP mRNA levels are displayed on the lower part of each panel. Band intensity were measuredon a phosphoimager, and the PACAP signal was normalized to the S16 internal control for each sample. Values shown are the range of twoindependent experiments along with the mean, indicated by bars. Hormone treatments are shown at the bottom.

5190 PR AND PACAP IN OVARY Endo • 1999Vol 140 • No 11

mature oocytes (35–39). One such local regulator is proges-terone. The importance of this steroid in successful ovulationis supported by a long list of experimental evidence dem-onstrating the direct stimulatory effect of progesterone on theovulation rate (5, 6). This intraovarian effect of progesteroneis thought to be PR dependent because RU486, a progester-one antagonist, inhibits ovulation at the ovarian level andmimics a clinical luteinizing unruptured follicle syndrome inhumans (40–42). Indeed, PRs are expressed in luteinizinggranulosa cells of preovulatory follicles of many species ex-amined thus far, during the narrow time window betweenthe preovulatory gonadotropin surge and ovulation (12–17,29). Ablation of these PRs by gene knock-out technologyresulted in animals with no gonadotropin-induced ovulationdespite normal expression of gonadotropin receptors (18, 19,43). Thus, the identification of PR downstream events shouldbe essential for understanding the molecular mechanismsunderlying successful ovulation and subsequent embryo-genesis. We have isolated the PACAP cDNA in the subtrac-tion cloning approach that was used to enrich progesterone-stimulated genes in luteinizing granulosa cells, providing

one of the first steps toward understanding functional rolesthat PRs play in mammalian ovulation.

PACAP, originally thought to be expressed in pituitarycells and to play a crucial role in pituitary hormone synthesis(44–45), is now known to be expressed and function in avariety of tissues (46). At the ovarian level in particular,PACAP mRNA and protein have been reported to be pre-dominantly expressed in luteinizing granulosa cells of pre-ovulatory follicles (33, 34) and, to a much lesser extent, bytheca-interstitial cells of follicles at all stages (34). It has alsobeen reported that luteal cells express PACAP proteins at allcyclic stages (34). Consistent with these previous results, wehave also observed predominant expression of PACAPmRNA to luteinizing granulosa cells of preovulatory folli-cles. However, we have observed little PACAP mRNA ex-pression in other cell types. This difference may be attrib-utable to the sensitivity of different in situ hybridizationapproaches. Alternatively, it may be due to differences incRNA probes corresponding to different regions of thePACAP mRNA. In fact, multiple transcripts, some of whichare alternatively spliced (47), encode PACAP. The PACAP

FIG. 6. PR mRNA expression precedes PACAP mRNA expression in vivo in PMSG-primed rat ovary. Immature rats at 22–23 days of age wereprimed 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 wereuntreated (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 ovarieswas used for RNA isolation and subsequent RT-PCR assays for PR and PACAP mRNA using 20 cycles of amplification (A); ribosomal proteinS16 mRNA was used as an internal control. Values shown are the range of the two groups of animals along with the mean, which is indicatedby bars. The other set of ovaries was used for in situ hybridization to localize PR and PACAP mRNAs on adjacent ovarian sections using35S-labeled antisense RNA probes synthesized from the rPR-1 (15) or the clone OKPS 9. After hybridization, tissue sections were exposed toKodak 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-condenserat a 1003 magnification (C).

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cDNA template used in this study is the subtracted cloneOKPS 9. This clone contains the 39-portion of the PACAPmRNA as expected from the cloning strategies. The 369 bpof this clone are identical to the corresponding region (2114–2483 bp) of the previously reported rat PACAP (GenBankaccession number M63006; Ref. 23). However, the most 39-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 alter-natively spliced exons. In this case, the clone OKPS 9 mustrepresent the PACAP isoform corresponding to the previ-ously reported mouse (31) and human (32) PACAP mRNAisoform.

Previous studies (33, 34) have demonstrated that gonad-otropins and forskolin induce PACAP mRNA in luteinizinggranulosa cells of preovulatory follicles in immature ratsprimed with gonadotropins. We have extended these studiesby demonstrating the direct stimulatory effect of hCG andforskolin in cultured granulosa cells. We have also demon-strated the close temporal correlation between the preovu-latory LH surge and PACAP mRNA in ovaries of adult ratsduring the preovulatory period, extending the previous re-sult showing transient expression of ovarian PACAP mRNAduring the early morning of estrus (34). In addition, ourresults demonstrate that PACAP gene expression occurslater than PR gene expression in luteinizing granulosa cells.Most importantly, our results demonstrate the inhibitoryeffect of ZK98299 on cAMP-induced PACAP expression.These results together identify PRs as one of the first se-quential links between the preovulatory LH surge andPACAP gene expression. Because both LH and forskolinstimulate PR synthesis at the level of transcription in theabsence of ongoing protein synthesis (Ref. 21 and this study),the inhibitory effect of cycloheximide on forskolin-inducedPACAP mRNA expression in cultured granulosa cells mustbe due, at least in part, to the lack of PR synthesis. ThePACAP promoter sequences available (48, 49) do not containconsensus PREs. Thus, the issue of whether PRs target thePACAP gene promoter directly or indirectly through othermolecules remains to be determined. The results presentedin this study do not exclude the possibility that more geneproducts may be involved between PRs and PACAP geneexpression. One reported gene that requires PR activation inbovine granulosa cells is oxytocin (50). Thus, it will be im-portant to elucidate whether oxytocin expression andPACAP expression are interdependent, leading to luteiniza-tion and/or ovulation.

Because the PR, a ligand-induced transcription factor, isindispensable for gonadotropin-induced ovulation and sub-sequent luteinization (18, 19, 43), the progesterone-stimu-lated gene PACAP must play a role in one or both of theseevents. Thus, the identification of PACAP as a progesterone-induced gene in luteinizing granulosa cells signifies an ex-citing window of new challenges. If PACAP should functionas an autocrine and/or paracrine modulator, mediating pro-gesterone action leading to ovulation and luteinization as wesuggest here, PACAP needs to be processed and secreted tobind its own receptors in its target cells. Although the closelink between PACAP synthesis and secretion has been dem-onstrated in cultured granulosa cells (51), the mechanismsunderlying PACAP secretion are poorly understood. It ispossible that PR activation also triggers cellular events lead-ing to PACAP secretion. Alternatively, the preovulatory go-nadotropin surge may independently trigger cellular eventsfor protein secretion. Despite the lack of understanding whatcontrols PACAP secretion, PACAP has been shown to affectseveral cellular events associated with ovulation. It stimu-lates intracellular cAMP accumulation and progesterone

FIG. 7. PR mRNA expression precedes PACAP mRNA expression invivo in adult rat ovary during the preovulatory period. Adult femalerats exhibiting at least two consecutive 4-day estrous cycles werekilled by decapitation. Trunk blood was collected for serum LH con-centrations as determined by LH RIA (the peak of the surge was seenat 1800 h). One set of ovaries was used for RNA isolation and sub-sequent RT-PCR assays for PR and PACAP mRNA using 20 cycles ofamplification (A); ribosomal protein S16 mRNA was used as an in-ternal control. Values shown are the range of two groups of animalsalong with the mean, indicated by bars. The other set of ovaries wasused for in situ hybridization to localize PR and PACAP mRNAs onadjacent ovarian sections using 35S-labeled antisense RNA probessynthesized from the rPR-1 (15) or the clone OKPS 9. After hybrid-ization, tissue sections were exposed to Kodak XAR-5 film (EastmanKodak Co.) for 2 days, and the film was directly scanned for inverseimages using the Nikon LS-1000 film scanner (B).

5192 PR AND PACAP IN OVARY Endo • 1999Vol 140 • No 11

production in granulosa cells (51, 52), presumably throughbinding to PACAP receptors. The issue of whether both typeI and type II receptors are present in granulosa cells of pre-ovulatory follicles and mediate the effect of PACAP on pro-gesterone synthesis has been addressed in previously pub-lished papers (53, 54) but still remains to be further examined.Nonetheless, it will be important to understand the relation-ship between PACAP and other local regulators such asproteases that have been implicated in ovulation (55, 56).Another aspect of ovulation that PACAP affects is meioticmaturation of oocytes (57, 58) presumably through its typeI receptor (58). Because the PACAP type I receptor is ex-pressed even in primordial germ cells (59), the LH-inducedproduction of PACAP should dictate resumption of meioticmaturation of dictyate oocytes. Taken together, these resultsdemonstrate that PR-induced PACAP synthesis may be acritical event for the initiation of gonadotropin-induced ovu-lation and oocyte meiotic maturation.

Acknowledgments

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

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