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Endocrinology Vol. 142, No. 9 3828-3835
Copyright © 2001 by The Endocrine Society

ARTICLES

Stage-Dependent Regulation of Ovarian Pituitary Adenylate Cyclase-Activating Polypeptide mRNA Levels by GnRH in Cultured Rat Granulosa Cells

Jy-Young Park1, Jeong-Ho Park, Hyun-Jeong Park, Jy-Young Lee, Yu-Il Lee, Keesook Lee and Sang-Young Chun

Hormone Research Center (J.-Y.P., J.-H.P., H.-J.P., K.L., S.-Y.C.) and Department of Obstetrics and Gynecology (J.-Y.L., Y.-I.L.), Chonnam National University, Kwangju 500-757, Republic of Korea

Address all correspondence and requests for reprints to: Dr. Sang-Young Chun, Hormone Research Center, Chonnam National University, Kwangju 500-757, Korea. E-mail: sychun{at}chonnam.chonnam.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study was designed to test whether GnRH regulates pituitary adenylate cyclase-activating polypeptide mRNA levels in a stage-dependent manner during follicle development in the rat ovary. The granulosa cells of preovulatory and immature follicles obtained from PMSG- and estrogen-treated rats, respectively, were cultured in serum-free conditions in the presence of various hormones. GnRH receptor mRNA expression was detected in both preovulatory and immature granulosa cells and was down-regulated by gonadotropins. Treatment of preovulatory granulosa cells with GnRH agonist stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner. In situ hybridization analysis of cultured preovulatory follicles revealed that GnRH-induced pituitary adenylate cyclase- activating polypeptide signals were detected in granulosa cells, but not thecal cells. In immature granulosa cells, cotreatment with GnRH agonist suppressed FSH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in a dose-dependent manner, whereas treatment with GnRH alone had no effect. Furthermore, treatment with GnRH antagonist inhibited LH-induced pituitary adenylate cyclase-activating polypeptide gene expression in preovulatory granulosa cells, whereas it stimulated FSH-induced pituitary adenylate cyclase-activating polypeptide gene expression in immature granulosa cells. Interestingly, GnRH-stimulated pituitary adenylate cyclase-activating polypeptide mRNA levels in preovulatory granulosa cells was inhibited by arachidonyltri fluoromethyl ketone, an inhibitor of phospholipase A2, but not by an inhibitor of protein kinase A or C. Lastly, treatment of preovulatory follicles with pituitary adenylate cyclase-activating polypeptide antagonist suppressed GnRH-stimulated progesterone production during 6–9 h of culture. Taken together, these results demonstrate the stage-dependent regulation of pituitary adenylate cyclase-activating polypeptide mRNA levels by GnRH, the stimulatory and inhibitory effect in granulosa cells of preovulatory and immature follicles, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH PLAYS A primary role in the control of reproductive functions in mammals. In addition to its well documented role in regulating the biosynthesis and secretion of the pituitary gonadotropins LH and FSH (1, 2), GnRH has been implicated as an autocrine/paracrine regulator in the ovary (3, 4). GnRH-like proteins that bind to the GnRH receptor (5) and GnRH gene transcripts (6, 7) have been demonstrated in the rat ovary. In situ hybridization experiments have shown that the GnRH mRNA is abundantly expressed in rat granulosa cells (8). Moreover, in addition to a GnRH-binding site (3, 4), the expression of the gene encoding the GnRH receptor in the rat ovary is also reported (9, 10, 11). Thus, the presence of an intrinsic GnRH system, complete with ligand, receptor, and biological responses, has been demonstrated in the rat ovary.

In the ovary the nature of the response to GnRH is largely related to the stage of follicle development (12). It is clear that the inhibitory effects of GnRH predominate in immature follicles that are responsive to gonadotropin stimulation. For example, in granulosa cells of small antral follicles, GnRH antagonizes the action of FSH on steroidogenesis and LH receptor concentrations in vitro (13, 14, 15) and causes apoptotic cell death of granulosa cells in vivo (16). In contrast, in preovulatory follicles, GnRH mimics certain physiological effects of LH, including induction of ovulation by stimulating the transcription of tissue-type plasminogen activator (17), PG endoperoxidase synthase type 2 (18), and PR (19) as well as initiation of meiotic maturation (20). The molecular basis for these different responses to GnRH remains to be determined.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide isolated from ovine hypothalamus (21), has recently been suggested to act as an autocrine/paracrine regulator in the ovary. PACAP mRNA is found in granulosa cells of preovulatory follicles after gonadotropin stimulation as well as in some thecal/interstitial cells in the rat ovary (22, 23). PACAP stimulates steroidogenesis and cAMP accumulation in cultured rat granulosa cells (24, 25). Furthermore, we recently reported the stage-dependent expression of PACAP type I receptor mRNA, which suggests a dual role of PACAP in immature and preovulatory follicles (26). This led us to postulate that ovarian GnRH might regulate PACAP mRNA levels in a stage-dependent manner during follicle development. We report here the stimulatory and inhibitory effects of GnRH on PACAP mRNA levels in granulosa cells of preovulatory and immature follicles, respectively. We also demonstrated a role for phospholipase A2 activation in GnRH-stimulated PACAP mRNA levels in preovulatory follicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hormones and reagents
Ovine LH (LH-S-26; 2300 IU/mg) and purified pituitary hFSH (ISIAFP-1; 8466 IU/mg) were obtained from the National Hormone and Pituitary Distribution Program, NIDDK, NIH (Baltimore, MD). Diethylstilbestrol (DES) and PMSG were purchased from Sigma (St. Louis, MO). GnRH antagonist (Org30850), [D-pGlu1,D-plu2,D-Trp3,6]GnRH, and a GnRH agonist, [D-Ala6-(N-3fMe)Leu7(Fug)]GnRH, were provided by Organon (Oss, The Netherlands). PACAP-(6–38), an antagonist of PACAP-38, and PACAP-(6–27), an antagonist of PACAP-27, were synthesized from PepTron (Daejeon, Korea). Arachidonyltri fluoromethyl ketone (AACOCF3) was purchased from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). MDL-12,330A (MDL) and chelerythrine (CE) chloride were purchased from Calbiochem (San Diego, CA).

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 of darkness, 14 h of light, with lights on from 0600–2000 h). The animals had ad libitum access to food and water. Animals were implanted with SILASTIC brand capsules (15 mm; Dow Corning Corp., Midland, MI) containing DES at 21–24 d of age to stimulate the development of multiple immature follicles. Three days later, the animals were killed by cervical dislocation, and the ovaries were removed for granulosa cell collection. Animals (26 d old, 60–65 g) were also injected sc with 10 IU PMSG to induce multiple growth of preovulatory follicles. Forty-eight to 52 h later, the animals were killed by cervical dislocation, and the ovaries were removed for follicle dissection or granulosa cell collection.

Culture of preovulatory follicles
Preovulatory follicles (>800 µm in diameter) were isolated by fine forceps from ovaries of immature rats collected 48–52 h after PMSG injection, and follicle culture was performed as previously described in a serum-free condition (23). Fifteen to 20 follicles were cultured in glass vials containing 400 µl MEM (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin, streptomycin, L-glutamine, and 0.1% BSA (wt/vol, fraction V, Sigma) in the absence or presence of different hormones. Cultures were maintained for up to 24 h at 37 C under 5% CO2-95% O2. After incubation, follicles were fixed for in situ hybridization analysis. In some cultures, media were collected for the determination of progesterone levels by RIA.

Granulosa cell isolation and culture
Granulosa cells of preovulatory and immature follicles were collected from ovaries of immature rats treated with PMSG for 2 d and with DES for 3 d, respectively, by the method of follicular puncture. Ovaries were incubated in DMEM/Ham’s F-12 supplemented with antibiotics and 0.1% BSA containing 0.5 M sucrose and 10 mM EGTA at 37 C for 30 min. Ovaries were washed three times in fresh DMEM/Ham’s F-12, and individual follicles were punctured using 23-gauge needles under a dissection microscope. Cells were counted using trypan blue and cultured at a density of 1 x 106 cells/60-mm dish in 2.5 ml DMEM/Ham’s F-12 supplemented with antibiotics and 0.1% BSA. Hormones were added at the beginning of culture, and cells were incubated at 37 C in a humidified 95% air-5% CO2 incubator. After incubation, cells were collected for RNA isolation.

Northern blot analysis
Total RNA from cultured granulosa cells was isolated using Tri-Reagent solution (Sigma). Twenty micrograms of total RNA were fractionated by electrophoresis on a 1.2% agarose gel containing formaldehyde and were transferred to nylon membranes by capillary blotting with 20 x SSC (sodium citrate-sodium chloride). After UV cross-linking and prehybridization, membranes were hybridized overnight at 42 C in a solution containing 50% formamide, 5 x SSC, 1.6 x Denhart’s solution, 1 mM EDTA, 250 µg/ml denatured herring sperm DNA, 500 µg/ml yeast transfer RNA, and a total of 2–4 x 106 cpm of a 32P-labeled rat PACAP (23) or GnRH receptor cDNA probe (27). After hybridization, membranes were washed twice for 5 min each time at room temperature in 2 x SSC and 0.1% SDS, followed by 1 h at 65 C in 0.5 x SSC and 0.1% SDS. Membranes were then exposed using Kodak RX film (Eastman Kodak Co., Rochester, NY) for 1 d at -80 C. For normalization of data, blots were stripped by boiling in 0.1 x SSC and 0.5% SDS twice for 20 min each time before reprobing with a cDNA probe for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). The band intensities were subsequently measured using a phosphorimager (Bio-Rad Laboratories, Inc., Hercules, CA), and the signals were normalized to the GAPDH internal control.

In situ hybridization analysis
Cultured preovulatory follicles 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)-coated microscope slides, fixed in 4% paraformaldehyde in PBS, and stored at -70 C until analyzed. The hybridization procedure was essentially the same as previously described (23). In brief, sections were pretreated serially with 0.2 M HCl, 2 x SSC, pronase E (0.125 mg/ml; Sigma), 4% paraformaldehyde, and acetic anhydride in triethanolamine. Hybridization was carried out at 52–55 C overnight in the mixture containing 35S-labeled rat PACAP cRNA probe (108 cpm/ml), 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, 1 x Denhardt’s solution, 10% dextran sulfate, 1 µg/ml carrier transfer RNA, and 10 mM dithiothreitol. Posthybridization 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 0.1 x SSC. Slides were dipped into NTB-2 emulsion (Eastman Kodak Co.) and exposed at 4 C until they were developed after 2 wk. The slides were stained with hematoxylin and eosin and examined under the light microscope with bright- and darkfield illumination.

RIA
Concentrations of progesterone in culture media were determined by RIA, as previously described (23), and expressed as nanograms per follicle.

Data analysis
Statistical differences were assessed by one-way ANOVA, followed by t test, and P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gonadotropin regulation of GnRH receptor mRNA levels
To determine the presence and regulation of GnRH receptor mRNA levels by gonadotropins in different stages of follicle development, total RNA extracted from cultured granulosa cells was analyzed by Northern blotting. Because physiological levels of LH and FSH are capable of stimulating preovulatory and immature follicles to complete their final differentiation, respectively (28), granulosa cells of preovulatory follicles were treated with LH, whereas those of immature follicles were treated with FSH. As shown in Fig. 1AGo, three different size transcripts were detected in granulosa cells of both preovulatory (left panel) and immature follicles (right panel) as well as in the pituitary. The levels of GnRH receptor mRNA were decreased by gonadotropin treatments. The most intense band had sizes of 5.0 and 4.5 kb. Quantitative analysis of GnRH receptor signals (Fig. 1BGo) showed a different pattern of regulation by gonadotropins. In granulosa cells of preovulatory follicles (left panel), LH treatment caused a gradual decrease in the levels of 4.5-kb GnRH receptor transcript (48% decrease at 12 h). The levels of 5.0-kb transcript showed a 50% decrease 6 h after treatment. In contrast, in granulosa cells of immature follicles (right panel), FSH treatment caused a rapid decrease in the levels of all transcripts (55–70% decrease at 3 h).



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  		Figure 1. Time-dependent expression of GnRH receptor mRNA by gonadotropins in cultured granulosa cells. A, Granulosa cells, obtained from ovaries of PMSG-primed (left panel) and DES-treated (right panel) immature rats were cultured in serum-free conditions at 37 C in the presence of 200 ng/ml LH or FSH for up to 12 h. Total RNA (20 µg/lane) was analyzed for GnRH receptor mRNA levels by Northern blotting using a rat GnRH receptor cDNA probe. The estimated sizes of GnRH receptor transcripts and the migration distance of 28S and 18S ribosomal RNA are indicated. The expression of GAPDH was used as an internal standard. Pit, Pituitary. B, Quantitation of the corresponding data in A. The different GnRH receptor transcripts were quantified using a phosphorimager, and data were normalized for GAPDH RNA levels in each sample. Results are expressed relative to levels of the 4.5-kb GnRH transcript found at 0 h after treatment. Each data point represents the mean ± SEM from two independently performed experiments.

 
Stage-dependent effect of GnRH agonist on PACAP mRNA levels
Gonadotropins have been shown to stimulate PACAP mRNA levels in granulosa cells of preovulatory follicles (23). To study the effect of GnRH agonist on PACAP mRNA levels, granulosa cells of preovulatory follicles obtained from ovaries of PMSG-primed rats were cultured in serum-free conditions. Northern blot analysis revealed that treatment with GnRH agonist stimulated the levels of PACAP mRNA in a dose-dependent manner (Fig. 2AGo). The PACAP mRNA levels stimulated by GnRH agonist were lower than those stimulated by LH. No additive effect of GnRH agonist and LH or FSH on PACAP mRNA levels was observed (data not shown). The levels of PACAP mRNA were 2.6-fold higher (P < 0.05) in cells treated with 10-7 M GnRH agonist than in cells treated with 10-10 M GnRH agonist (Fig. 2BGo). To determine the cell types expressing PACAP mRNA in GnRH agonist-treated preovulatory follicles, in situ hybridization was performed on sections of follicles incubated in the presence of GnRH agonist for 6 h. PACAP signals were detected in granulosa cells, but not thecal cells, of preovulatory follicles. (Fig. 2CGo, a and b). Under higher magnification, evenly distributed signals of PACAP mRNA were seen in the granulosa cells (Fig. 2CGo, c). No specific signal was detected in follicles hybridized with sense probe (data not shown).



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  		Figure 2. Effect of GnRH agonist on PACAP mRNA expression in granulosa cells of preovulatory follicles. A, Granulosa cells of preovulatory follicles were cultured in serum-free conditions in the absence (control; C) or presence of LH (200 ng/ml) and increasing doses of GnRH agonist (GnRH A) for 6 h. Total RNA (10 µg/lane) was analyzed for PACAP mRNA levels by Northern blotting using a rat PACAP cDNA probe. The estimated sizes of PACAP transcripts and the migration distance of 28S and 18S ribosomal RNA are indicated. The expression of GAPDH was used as an internal standard. B, Quantitation of the data in A. The 3-kb PACAP transcript was quantified using a phosphorimager, and data were normalized for GAPDH RNA levels in each sample. Results are expressed relative to PACAP mRNA levels found in follicles treated with 10-10 M GnRH agonist. Each data point represents the mean ± SEM from four independently performed experiments. C, Localization of PACAP mRNA after treatment with GnRH agonist. Sections of preovulatory follicles cultured in the presence of GnRH agonist (10-7 M) for 6 h were hybridized with a rat PACAP cRNA probe. Note the presence of specific signals in granulosa cells (Gc), but not in thecal cells (Tc). Photomicrographs were taken under brightfield (a and c) and darkfield (b) illumination. Magnification: a and b, x100; c, x400.

 
FSH has been shown to induce PACAP gene expression in preovulatory follicles (23). To determine whether FSH also induces PACAP gene expression in immature follicles, granulosa cells collected from ovaries of DES-treated rats were incubated in serum-free conditions in the presence of FSH for up to 24 h. Northern blot analysis revealed that FSH treatment resulted in a transient stimulation of PACAP mRNA levels, reaching maximum levels 3–6 h after treatment (Fig. 3AGo). To study the stage-dependent effect of GnRH agonist on PACAP mRNA levels, granulosa cells of immature follicles were cultured in the presence of FSH and GnRH agonist for 6 h. In contrast to the stimulatory effect of GnRH agonist in granulosa cells of preovulatory follicles (Fig. 2AGo), treatment of granulosa cells of immature follicles with GnRH agonist did not stimulate PACAP mRNA levels (Fig. 3BGo). Moreover, cotreatment with GnRH agonist suppressed FSH-stimulated PACAP mRNA levels in a dose-dependent manner. Quantitative analysis (Fig. 3CGo) showed a 75% decrease (P < 0.05) in FSH-stimulated PACAP mRNA levels by treatment with 10-7 M GnRH agonist.



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  		Figure 3. Effect of GnRH agonist on FSH-stimulated PACAP mRNA expression in granulosa cells of immature follicles. A, Granulosa cells, obtained from ovaries of DES-treated immature rats, were cultured in serum-free conditions in the presence of FSH (200 ng/ml) for up to 24 h. B, Granulosa cells were cultured in the absence (control; C) or presence of FSH with or without increasing doses of GnRH agonist (GnRH A) for 6 h. Total RNA (10 µg/lane) was analyzed for PACAP mRNA levels by Northern blotting using a rat PACAP cDNA probe. The estimated sizes of PACAP transcripts and the migration distance of 28S and 18S ribosomal RNA are indicated. The expression of GAPDH was used as an internal standard. C, Quantitation of the data in B. The 3-kb PACAP transcript was quantified using a phosphorimager, and data were normalized for GAPDH RNA levels in each sample. Results are expressed relative to PACAP mRNA levels found in granulosa cells treated with FSH alone. Each data point represents the mean ± SEM from two independently performed experiments.

 
Stage-dependent effect of GnRH antagonist on gonadotropin-stimulated PACAP mRNA levels
As GnRH is expressed in the rat ovary (5, 6, 7), we determined whether the stimulatory action of gonadotropins on PACAP mRNA levels is mediated via the action of endogenous GnRH. In granulosa cells of preovulatory follicles (Fig. 4AGo, left panel), LH-induced PACAP gene expression was inhibited by cotreatment with GnRH antagonist in a dose-dependent manner. FSH-induced PACAP gene expression was also inhibited by cotreatment with GnRH antagonist (data not shown), ruling out the possibility that the inhibitory effect of GnRH antagonist may be dependent on the different type of gonadotropins. In contrast, in granulosa cells of immature follicles (Fig. 4AGo, right panel), cotreatment with GnRH antagonist enhanced the stimulatory effect of FSH on PACAP mRNA expression in a dose-dependent manner. Treatment with GnRH antagonist alone had no effect. Quantitative analysis (Fig. 4BGo) showed a 70% decrease and a 1.6-fold increase (P < 0.05) in LH- and FSH-stimulated PACAP mRNA levels in granulosa cells of preovulatory and immature follicles, respectively, by treatment with 10-5 M GnRH antagonist.



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  		Figure 4. Effect of GnRH antagonist on gonadotropin-stimulated PACAP mRNA expression in cultured granulosa cells. A, Granulosa cells, obtained from ovaries of PMSG-primed (left panel) and DES-treated immature rats (right panel) were cultured in serum-free conditions in the absence (control; C) or presence of LH (200 ng/ml) and FSH (200 ng/ml) with or without increasing doses of GnRH antagonist (GnRH Ant) for 6 h. Total RNA (10 µg/lane) was analyzed for PACAP mRNA levels by Northern blotting using a rat PACAP cDNA probe. The estimated sizes of PACAP transcripts and the migration distances of 28S and 18S ribosomal RNA are indicated. The expression of GAPDH was used as an internal standard. B, Quantitation of the corresponding data in A. The 3-kb PACAP transcript was quantified using a phosphorimager, and data were normalized for GAPDH RNA levels in each sample. Results are expressed relative to PACAP mRNA levels found in granulosa cells treated with LH or FSH alone. Each data point represents the mean ± SEM from three or four independently performed experiments.

 
Role of phospholipase A2 activation in GnRH-stimulated PACAP mRNA levels
In addition to the stimulation of the protein kinase C pathway, the actions of GnRH are mediated by other intracellular messengers, such as those derived from the adenylate cyclase and phospholipase A2 pathways (4). To determine the signaling pathway for PACAP mRNA induction by GnRH, granulosa cells of preovulatory follicles were incubated for 6 h in the presence of increasing doses of AACOCF3, an inhibitor of phospholipase A2; 10 µM CE, an inhibitor of PKC; or 10 µM MDL, an inhibitor of protein kinase A, with or without GnRH agonist (10-7 M). As shown in Fig. 5AGo, inclusion of AACOCF3 decreased GnRH-stimulated PACAP mRNA levels in a dose-dependent manner. Treatment with 100 µM AACOCF3 caused a 75% decrease (P < 0.05) in GnRH agonist-stimulated PACAP mRNA levels (Fig. 5BGo). In contrast, the addition of CE or MDL resulted in a minimal effect on GnRH agonist-stimulated PACAP mRNA levels.



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  		Figure 5. Effect of PLA2 activation on GnRH-stimulated PACAP mRNA expression in granulosa cells of preovulatory follicles. A, Granulosa cells of preovulatory follicles were incubated in serum-free conditions in the absence (control; C) or presence of GnRH agonist (10-7 M) with or without CE (10 µM), MDL (10 µM) or increasing doses of AACOCF3 for 6 h. Total RNA (10 µg/lane) was analyzed for PACAP mRNA levels by Northern blotting using a rat PACAP cDNA probe. The estimated sizes of PACAP transcripts and the migration distance of 28S and 18S ribosomal RNA are indicated. The expression of GAPDH was used as an internal standard. B, Quantitation of the data in A. The 3-kb PACAP transcript was quantified using a phosphorimager, and data were normalized for GAPDH RNA levels in each sample. Results are expressed relative to PACAP mRNA levels found in granulosa cells treated with GnRH agonist alone. Each data point represents the mean ± SEM from two independently performed experiments.

 
Suppression by PACAP antagonist of GnRH-stimulated progesterone production by cultured preovulatory follicles
Because GnRH is known to stimulate progesterone production (18) and PACAP mRNA levels in preovulatory follicles (Fig. 2Go), we further tested whether the stimulatory action of GnRH on progesterone production is mediated via the action of endogenous PACAP. Treatment of preovulatory follicles with 10-7 M GnRH agonist resulted in a gradual increase in progesterone production during the 24-h culture (Fig. 6Go). The addition of PACAP-38 or -27 antagonist partially inhibited the GnRH action on the stimulation of progesterone production during the 6- to 9-h culture.



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  		Figure 6. Effect of PACAP antagonist on GnRH-stimulated progesterone production by cultured preovulatory follicles. Preovulatory follicles were cultured in the presence of GnRH agonist (10-7 M) with or without PACAP-38 or -27 antagonist (Ant; 10-5 M) for up to 24 h. The progesterone concentration in culture medium was determined by RIA. Each data point represents the mean ± SEM from six independently performed experiments. *, P < 0.05 compared with the respective value found in follicles treated with GnRH agonist alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these studies document that GnRH regulates PACAP mRNA levels in a stage-dependent manner. In cultured granulosa cells of immature follicles, GnRH had an inhibitory effect on PACAP mRNA levels. This idea is supported by the present data showing the inhibition and enhancement of FSH-induced PACAP gene expression by GnRH agonist and antagonist, respectively. An abundance of evidence has been accumulating to document that the inhibitory effects of GnRH is largely restricted to immature follicles under the gonadotropin stimulation including inhibition of FSH-stimulated steroidogenesis and LH receptor concentrations (13, 15). In cultured granulosa cells of preovulatory follicles, however, GnRH had a stimulatory effect on PACAP mRNA levels. GnRH agonist itself could stimulate PACAP mRNA levels. Furthermore, GnRH antagonist suppressed LH-induced PACAP gene expression. Several lines of evidence have been reported to support the stimulatory effects of GnRH in preovulatory follicles, including induction of ovulation and oocyte maturation (20). Because the present data demonstrated the presence of GnRH receptor in cultured granulosa cells, these GnRH actions on PACAP mRNA levels might be exerted through specific binding to its receptor. Interestingly, treatment with GnRH agonist induced PACAP gene expression in preovulatory follicles by activating the phospholipase A2 pathway. In addition to the PKC pathway, GnRH has been shown to stimulate the phospholipase A2 pathway in the ovary (29) and pituitary (30). The present results further indicate the ability of GnRH to inhibit FSH action in immature follicles and to mimic some, but not all, of the actions of LH in preovulatory follicles.

The present study demonstrates that three GnRH receptor mRNAs with sizes of 5.0, 4.5, and 1.6 kb were detectable in cultured granulosa cells and that the levels of transcripts are regulated by gonadotropins. These findings add further support to the hypothesis of a potential role of GnRH as an ovarian local factor involved in the regulation of ovarian function. A similar observation showing GnRH receptor transcripts different in size was reported in the pituitary and ovary (31, 32). The nature of these different GnRH receptor mRNA species is unknown at present; they may be the products of alternative transcriptional initiation and termination and/or alternative splicing. Furthermore, we found that treatment with exogenous gonadotropins results in a down-regulation of GnRH receptor transcripts with a more rapid decrease by FSH than by LH. The down-regulation of GnRH receptor gene expression by LH/hCG has been detected by RT-PCR in cultured preovulatory rat granulosa cells (10) and in the ovary (33). GnRH receptor transcripts remained elevated for up to 3–6 h in granulosa cells of preovulatory follicles cultured with LH, compared with a decline within 1 h in granulosa cells of immature follicles cultured with FSH. This different effects of gonadotropins on GnRH receptor down-regulation may be explained by differences due to temporally induced alterations in cell phenotypes (preovulatory vs. immature) as well as differences in the type of gonadotropin (LH vs. FSH).

As GnRH antagonizes the action of FSH that promotes follicle development, it has been suggested that GnRH may act primarily as an atretogenic factor for the majority of follicles that do not ovulate (15). Support for this concept comes from studies showing that GnRH directly promotes apoptotic cell death in the ovary (16). In the present study treatment of immature granulosa cells with GnRH agonist antagonized the FSH action on PACAP mRNA levels. Because PACAP stimulates steroidogenesis and cAMP accumulation in cultured immature granulosa cells (24, 25) and promotes the growth of preantral follicles in vitro (unpublished observation), the action of FSH on the promotion of follicle development may be partially mediated by the endogenous production of PACAP. Hypothetically, the atretogenic action of GnRH might, therefore, be exerted by the inhibition of FSH-induced PACAP production. The present observation demonstrating the potentiation of FSH action on PACAP mRNA levels by cotreatment with GnRH antagonist further supports this concept. However, such a hypothesis needs to be further clarified by experimental data.

In contrast to the inhibitory effect of GnRH in immature follicles, GnRH had a stimulatory effect on PACAP mRNA levels in preovulatory follicles. GnRH has been shown to cause follicle rupture and oocyte maturation (20, 34) and to induce in vitro luteinization of preovulatory granulosa cells (35). Because our previous data (23, 26) and other recent studies (22, 36, 37) demonstrate a possible role for PACAP during the ovulatory process, the stimulatory action of GnRH on ovulation may be partially exerted by the increased production of PACAP. The present data showing the inhibition of GnRH-stimulated progesterone production by PACAP antagonist during 6–9 h culture further suggest a mediator role of PACAP in GnRH-induced luteinization. The lack of inhibition by PACAP antagonist of GnRH-stimulated progesterone production during 12–24 h culture may reflect the transient production of endogenous PACAP by GnRH. Indeed, a recent report demonstrates a role for PACAP during LH/hCG-induced periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells (36). In rat preovulatory follicles, it has been shown that GnRH has direct effect on progesterone production (38, 39). Although changes in GnRH mRNA levels are seen during postnatal development and luteinization (40), gonadotropin regulation of ovarian GnRH expression is largely unknown. The present observation showing a partial inhibition of LH-induced PACAP gene expression by GnRH antagonist suggests that LH may increase the endogenous expression of GnRH, and in turn, GnRH may stimulate ovulation by increasing PACAP production.

Interestingly, the GnRH signal for the induction of PACAP gene expression in preovulatory follicles was mediated through the phospholipase A2 (PLA2) signaling pathway, rather than the protein kinase A or PKC signaling pathway. In rat granulosa cells of immature follicles, PKC activation mediates GnRH action on FSH-induced steroidogenesis (41, 42) and on stimulation of IGF-binding protein-4 (43). However, PKC may not be the exclusive pathway by which GnRH exerts its effects. In preovulatory follicles, GnRH has been shown to cause a small increase in cAMP and to induce PG H synthase by the activation of tyrosine kinases, but not PKC (18). Furthermore, stimulation of the PLA2 pathway by GnRH has been reported in cultured rat luteal (44) and granulosa cells (29, 45). It has thus been proposed that there may be independent coupling of the GnRH receptor to phospholipase C and PLA2, possibly via different receptor subclasses (4). Support for this concept comes from recent studies demonstrating the identification of three distinct types of GnRH receptor in the bullfrog (46).

In summary, the present study has demonstrated the stimulatory and inhibitory effects of GnRH in preovulatory and immature follicles, respectively, on PACAP mRNA levels. GnRH action on the induction of PACAP gene expression in preovulatory follicles requires the activation of PLA2 pathway. Furthermore, PACAP partially mediates GnRH stimulation of preovulatory progesterone production. The precise mechanisms by which GnRH mediates differential effects on PACAP mRNA levels in granulosa cells at defined stages of follicle development need to be clarified.


   Acknowledgments
 
We thank the National Hormone and Pituitary Distribution Program (NIDDK, NIH) for the oLH and hFSH preparations.


   Footnotes
 
This work was supported by Korea Research Foundation Grant (KRF-99-015-DP0361) and HRC-98k1-0405, Republic of Korea (to S.Y.C.).

1 Present address: Infertility Medical Center of CHA and Eun General Hospital of Kwangju, Pochon CHA University, Seoul, Republic of Korea. Back

Abbreviations: AACOCF3, Arachidonyltri fluoromethyl ketone; CE, chelerythrine; DES, diethylstilbestrol; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; MDL, MDL-12,330A; PACAP, pituitary adenylate cyclase-activating polypeptide; PLA2, phospholipase A2.

Received March 19, 2001.

Accepted for publication May 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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