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Reproduction (2007) 134 281-292
DOI: 10.1530/REP-07-0051
Copyright © 2007 Society for Reproduction and Fertility
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RESEARCH

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary

Marzia Barberi, Barbara Muciaccia, Maria Beatrice Morelli, Mario Stefanini, Sandra Cecconi1 and Rita Canipari

Department of Histology and Medical Embriology, ‘La Sapienza’ University of Rome, Via A. Scarpa, 14, Rome 00161, Italy and 1 Department of Biomedical Sciences and Technologies, University of L’Aquila, L’ Aquila 67010, Italy

Correspondence should be addressed to R Canipari; Email: rita.canipari{at}uniroma1.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The bioactive pituitary adenylate cyclase-activating polypeptide (PACAP) is an extremely well-preserved regulatory peptide originally isolated from ovine hypothalamus. It exists in two forms, PACAP-27 and PACAP-38, which share the same N-terminal 27 amino acid residues and are derived from tissue-specific proteolytic processing of the 176-amino acid precursor protein (Arimura 1992a). The name reflects its potent stimulation of cAMP production in anterior pituitary cells (Miyata et al. 1989). On the basis of sequence similarity, PACAP belongs to the secretin–glucagon–vasoactive intestinal polypeptide (VIP) family of peptides (Kimura et al. 1990). The biological effects of PACAP are mediated through its binding to at least three types of G-protein-coupled seven transmembrane receptors. PACAP type I receptor (PAC1-R) binds PACAP-27 and PACAP-38 with a high affinity and VIP with a lower affinity; VPAC1-R and VPAC2-R bind PACAP and VIP with an equal affinity (Lutz et al. 1993). PAC1-R exists in variant forms generated by different splicing: a short form, a very short form due to the deletion of a 21-amino acid cassette in the N-terminal extracellular domain (Pantaloni et al. 1996), and five variants that either contain or lack each of the two alternative exons at the terminal end of the third intracellular loop of the receptor (Spengler et al. 1993).

PACAPs, VIP and their receptors are believed to play a role not only in the central nervous system, but also in various organs and peripheral tissues such as lung, testis, adrenal gland and ovary (Gottschall et al. 1990, Arimura 1992a, 1992b, 1998, Sherwood et al. 2000).

There is growing evidence that PACAP plays an important role in the female (Scaldaferri et al. 1996) reproductive system by acting as a potential local regulator of ovarian physiology. In the rat ovary, where PACAP and PAC1-R (Gras et al. 1996, Scaldaferri et al. 1996, Koh et al. 2000, Park et al. 2000, Vaccari et al. 2006) are transiently produced by gonadotrophin-stimulated preovulatory follicles, PACAP stimulates various ovarian functions, including cAMP accumulation, steroidogenesis and plasminogen activator (PA) production in cultured granulosa cells (Zhong & Kasson 1994, Gras et al. 1999, Apa et al. 2002). In addition, it accelerates meiotic maturation in rat cumulus-enclosed oocytes (Apa et al. 1997) and inhibits apoptosis in preovulatory follicles (Lee et al. 1999, Vaccari et al. 2006).

To further characterise the PACAP/receptor system in mouse ovary, this study aimed to verify the presence of this peptide and its receptor transcripts in mouse preovulatory follicles at the mRNA and protein levels and to demonstrate the presence of functional receptors on granulosa cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Animals
Immature CD1 mice (Charles River, Como, Italy) were housed under controlled temperature (25 °C) and light (12 h light/day) conditions with ad libitum access to food and water. Animals were maintained in accordance with the Italian Department of Health Guide for Care and Use of Laboratory Animals. Experimental protocols were approved by the ‘La Sapienza’ University Committee for Animal Care and Use. Mice were killed by cervical dislocation. Twenty-two-day-old immature mice were either killed or injected i.p. with 7 i.u. equine chorionic gonadotrophin (eCG, Intervet, Milan, Italy) to enhance multiple follicular development. After 46–48 h, the animals were killed, or injected with 5 i.u. human chorionic gonadotrophin (hCG, Intervet) and killed at different times after injection.

Tissue and cell isolation
Ovaries were removed aseptically and freed from adherent tissues in Hank’s balanced salt solution (HBSS, Gibco, Invitrogen). Whole ovaries were processed immediately for RNA or protein isolation.

Granulosa cells (GC) were obtained as described previously (Vaccari et al. 2006). Briefly, the largest follicles from each ovary were punctured with a 25-gauge needle and gently pressed to release GC. Cells were centrifuged at 250 g for 5 min and either resuspended in lysis buffer for RNA extraction or cultured at a density of 1.5 x 105/200 µl in Dulbecco’s modified Eagle medium (DMEM, Gibco, Invitrogen) supplemented with 1 mg/ml BSA, 2 mM glutamine and antibiotics (100 mM penicillin, 100 µg/ml streptomycin). The residual ovarian tissue, mainly theca/interstitial cells and granulosa cells from small preantral follicles, was homogenised in lysis buffer for RNA extraction.

Preovulatory follicles were isolated from ovaries collected 46–48 h after eCG injection in HBSS supplemented with 1 mg/ml BSA and cultured as described previously (Lee et al. 1999). Briefly, four follicles were cultured in polypropylene culture tubes containing 500 µl DMEM supplemented with 2 mM glutamine and antibiotics (penicillin, streptomycin) and 1 mg/ml BSA in the absence or presence of 100 ng/ml luteinising hormone (LH), or increasing concentrations of PACAP or VIP (10–9–10–7 M; Calbiochem/Merck Darmstadt).

In additional experiments, follicles were incubated with 10–7 M PACAP or VIP in the presence of 5 x 10–7 M PACAP and VIP antagonists, which were PACAP-38 (6–38) trifluoroacetate salt, the hybrid of neurotensin (6–11) and VIP (7–28; Bachem AG, Bubendorf, Switzerland), and the VIP receptor antagonist (D-P-chloro-Phe6,Leu17)-VIP (Sigma–Aldrich).

Morphological analysis of granulosa cell apoptosis
To evaluate the effect of PACAP and VIP on granulosa cell apoptosis, follicles were mechanically dissected from eCG-treated mice and GCs were released in the medium immediately before or after 24 h of follicle culture. Cells from single follicles were fixed for 15 min in 3% (w/v) paraformaldehyde/PBS and cytocentrifuged onto a glass slide at 200 g for 10 min. The samples were washed thrice with PBS and the chromatin was stained using the TUNEL (TdT-mediated dUTP-X nick end labelling) method according to the manufacturer’s instructions (Mebstain Apoptosis Kit Direct, MBL International, Woburn, MA, USA). Apoptotic cells were identified and counted in three or more randomly selected fields with at least 100 cells each.

RNA extraction and RT
Total RNA from whole ovaries, GC and residual ovarian tissue was isolated by a silica gel-based membrane spin column (RNeasy Kit, Qiagen S.p.A.). The purity and integrity of the RNA was checked spectroscopically and by gel electrophoresis. Total RNA (1 µg) was reverse-transcribed in a final volume of 20 µl, using the M-MLV Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s instructions.

Multiplex PCR
To determine the presence of PACAP and its receptor transcripts, a duplex-touchdown-PCR was performed. The reactions were carried out using a Multiplex PCR Kit (Qiagen) according to the manufacturer’s instruction, with the housekeeping gene ß-actin as internal control. To increase the specificity and the quality of target products, a touchdown PCR was performed. The initially high annealing temperature (Ta, 67 °C for PACAP and all the receptors, and 62 °C for VIP) was lowered by 1 °C per cycle to a ‘touchdown’ temperature of 59 °C for PACAP and all the receptors and 55 °C for VIP. This ‘touchdown’ temperature remained the same for the remaining 22 cycles. The primer sequences chosen are shown in Table 1Go. Each primer pair was previously tested alone for specific amplification. Primers for PAC1-R were chosen in a region that allowed the detection of all splice variants. For each sample, 10 µl PCR product was then subjected to electrophoresis on 2% (w/v) agarose gel and stained with ethidium bromide. The densitometric evaluation of the bands was performed with AIDA software (Advanced Image Data Analyzer 2.11 raytest GmbH, Straubenhartd, Germany). The relative mRNA levels were normalised against the expression of the housekeeping gene. The ß-actin primer set 1 was used for the normalisation of PACAP, VIP, PAC1-R and VPAC2-R mRNA levels, while the ß-actin primer set 2 was used for VPAC1-R. DNA contamination controls were performed using gene-specific primers on RNA without reverse transcriptase treatment. PCR products were sequenced to verify the specificity of amplified DNA.


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  		Table 1 Sequence of oligonucleotides used as RT-PCR primers.
 
Analysis of PAC1-R isoforms
To identify the PAC1-R splice variants present in the ovary, specific primer pairs (Table 1Go) were designed on the basis of the analysis of the PAC1-R mouse mRNA sequence obtained from NCBI GenBank. The forward (Fw) and reverse (Rv) hip/hop primers, which flanked the site of insertion of the hip/hop cassette region, were used to amplify all the isoforms in this region, whereas the hop-Rv-specific primer was located inside the hop cassette (Fig. 1Go). RT-PCR was performed with a Ta of 57 °C for 30 cycles. Following amplification, PCR products were purified with the SureClean Kit (Bioline GmbH, Luckenwalde, Germany). Twenty microlitres of each purified cDNA were digested with PvuII and/or AvaI restriction enzymes (Promega) for 4 h at 37 °C and the products of digestion were visualised by electrophoresis on 2% (w/v) agarose gel.


Figure 1
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  		Figure 1 Schematic diagram of the PAC1-R mRNA, based on the mouse or rat sequence, along with the primer locations.

 
Immunofluorescence
Freshly isolated ovaries from eCG- and hCG-stimulated mice were embedded in optimal tissue freezing medium (TBS, Duram, NC, USA), snap-frozen in liquid nitrogen and stored at 380 °C until being sectioned with a Leitz cryostat. Sections (7 µm) were fixed in 4% paraformaldehyde and permeabilised with 0.1% (v/v) Triton X-100. The sections were blocked for 3 h in 10% (v/v) normal goat serum (Sigma–Aldrich) to minimise non-specific binding, and incubated overnight at 4 °C with 1:1000 rabbit polyclonal anti-PACAP antiserum (kindly provided by Dr A Arimura; Koves et al. 1990). The sections were then incubated for 1 h at RT with 1:1000 Alexa Fluor 488-conjugated mouse anti-rabbit secondary antibody (Molecular Probes, Invitrogen) and observed under a fluorescence microscope. In control samples, the primary antibody was substituted with rabbit pre-immune serum.

In situ hybridisation histochemistry
Freshly isolated ovaries from eCG- and hCG-stimulated mice were fixed in Bouin’s fluid for 48 h at room temperature. Fixed ovarian tissue was embedded in paraffin and sectioned at 7 µm. Paraffin sections were mounted on microscope polylysine slides (Menzel-Glaser, Braunschweig, Germany), deparaffined and rehydrated. Slides were then post-fixed in 4% para-formaldehyde for 10 min at room temperature, and treated with 10 µg/ml proteinase K (Roche Diagnostics S.p.A.). Hybridisation was carried out overnight at 55 °C in a humidified chamber in a mixture containing 50% (v/v) formamide, 1 x standard sodium citrate (SSC), 1 x Denhardt’s solution, 10% (w/v) dextran sulphate, 200 mg/ml salmon sperm DNA and 300 ng/ml digoxigenin-labelled DNA PACAP probe. The probe was labelled by direct incorporation of DIG-dUTP (Roche Diagnostics) during PCR amplification performed with the same primers as those used to amplify PACAP in multiplex RT-PCR (PAC1-R, see Table 1Go). After hybridisation, washings were performed under stringent conditions to a final concentration of 0.1% (v/v) SSC. Sections were then incubated in blocking solution (100 mM Tris–HCl (pH 7.5), 100 mM NaCl, 2 mM MgCl2, 1% (w/v) BSA) and in 1:300 alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics) diluted in blocking solution. Colorimetric detection was developed with a chromogen substrate (NBT, BCIP; Roche Diagnostics). The sections were observed by light microscopy and not counterstained.

A digoxigenin-labelled ß-actin DNA probe was used as a positive control, whereas an unlabelled PACAP DNA probe, as well as competing hybridisations, with different mixtures (1:5 and 1:10) of digoxigenin-labelled/-unlabelled PACAP DNA probes, were used as negative and specificity control reactions.

Western blot analysis
Ovaries from untreated 22-day-old immature mice or from eCG-treated mice were lysed with RIPA buffer (10 mM Tris (pH 7.2), 150 mM NaCl, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 mM EDTA) containing protease inhibitors (Sigma–Aldrich) and sonicated on ice. The sonicated tissue was centrifuged for 2 min at 15 000 g at 4 °C and the supernatant was stored at –20 °C until use. Protein concentration was measured by BCA protein assay (Pierce Biotechnology, Rockford, IL, USA). ~50 µg proteins were subjected to SDS-PAGE under denaturing conditions on a 12% (w/v) acrylamide gel (Laemmli 1970) and transferred onto a nitrocellulose membrane (Schleicher & Schuell, Whatman GmbH, Dassel, Germany). Non-specific binding was blocked by incubation with 5% (w/v) low-fat dry milk and 0.3% (v/v) tween-20 in PBS calcium-magnesium-free (CMF). The membrane was then incubated overnight at 4 °C with rabbit anti-VPAC1-R polyclonal antibody (1:200, sc-30019; Santa Cruz Biotechnology, Heidelberg, Germany) and with a mouse monoclonal antibody anti-tubulin (1:1000, T5168, Sigma–Aldrich) in CMF added with 5% (w/v) low-fat dry milk. The membrane was washed thrice with CMF for 20 min at room temperature and then incubated with the AP-conjugated secondary anti-rabbit antibody (1:2000, Zymed, San Francisco, CA, USA) for 1 h at room temperature or anti-mouse biotinilated antibody (1:5000; Dako Italia S.p.A. Milano, Italy) followed by incubation with streptavidin–AP complex (1:1000, Dako) at room temperature for 1 h. After washing with washing buffer (0.1 M Tris (pH 9.5), 0.1 M NaCl), immunocomplexes were detected by western blot chemiluminescence reagent (CDP-star; NEN, Boston, MA, USA), following the manufacturer’s instructions. Molecular masses were measured by prestained molecular markers (Gibco).

Statistical analysis
Data are expressed as the mean ± S.E.M. from at least three independent experiments. Statistical analysis was performed using ANOVA followed by the Tukey–Kramer test for comparisons of multiple groups. Values with P<0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Expression of PACAP
To verify the presence of PACAP transcripts and characterise their expression pattern following gonadotrophin stimulation, total RNA was extracted from ovaries obtained from 14- and 22-day-old untreated mice, eCG-primed or eCG/hCG-primed mice. Furthermore, the level of specific mRNAs was assessed by multiplex RT-PCR.

Our data did not show PACAP expression in 14-day-old ovaries (data not shown), in untreated (22d) or eCG-treated whole ovaries from 22-day-old animals; PACAP expression was transiently induced by hCG treatment, when it became detectable after 1 h, reached a maximum level after 3–6 h, and decreased to unstimulated levels after 9 h (Fig. 2AGo). VIP transcripts were never observed at the time points considered (data not shown).


Figure 2
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  		Figure 2 Effect of gonadotrophin stimulation on PACAP gene expression detected by multiplex RT-PCR. Ovaries were collected from untreated 22-day-old immature mice (22d), 48 h after eCG stimulation (0) or at different times after hCG treatment. Total RNA, extracted from whole ovaries (A), granulosa cells (B) and residual ovarian tissue (C), was analysed separately and subjected to multiplex RT-PCR using primers specific for PACAP, as well as for ß-actin (primer set 1; Table 1Go) used as an internal control. An aliquot of each PCR product was electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. Expression levels were quantified by densitometric evaluation of the bands with a chemiluminescence detection system (raytest). PACAP values were normalised by their respective ß-actin values and represent the mean ± S.E.M. of three independent experiments carried out on separate animals. *P<0.05; **P<0.01; ***P<0.001 versus 22d.

 
To investigate the expression of PACAP in the different ovarian compartments, RNA was extracted from isolated GCs and residual ovarian tissue (TI cells) obtained from ovaries of 22-day-old untreated, eCG- and hCG-treated mice. As shown in Fig. 2BGo, the expression pattern of PACAP transcripts in isolated GCs mimicked that of whole ovary, with no expression before hCG treatment and transient expression between 1 and 6 h after gonadotrophin stimulation. Residual ovarian tissue displayed very low overall PACAP expression, which slightly, though significantly, increased after hCG injection (Fig. 2CGo), thus suggesting that, following gonadotrophin stimulation, PACAP was predominantly expressed in GCs.

Localisation of PACAP
To determine the cell type that expresses PACAP mRNA, in situ hybridisation histochemistry was performed on ovarian sections obtained from mice before and 6 h after hCG injection. The PACAP signal was absent in ovaries of eCG-treated mice (Fig. 3BGo); it was instead detected mainly in GCs of large preovulatory follicles after gonadotrophin stimulation (Fig. 3CGo). PACAP immuno-reactivity was very low in the ovaries of eCG-treated animals (Fig. 4AGo), whereas was observed in GCs and cumulus cells of preovulatory follicles 6 h after hCG stimulation (Fig. 4BGo).


Figure 3
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  		Figure 3 In situ hybridisation analysis of PACAP mRNA expression in mouse ovary. Ovarian sections were hybridised with DIG-dUTP-labelled cDNA probes generated by PCR, as described in Materials and Methods. No PACAP mRNA signals were detected in eCG-treated ovaries (B), whereas expression was clearly detected in GCs of large preovulatory follicles 6 h after hCG treatment (C, arrows). As a negative control, samples from hCG-treated ovaries at 6 h were hybridised in the presence of a 1:10 digoxigenin-labelled/-unlabelled PACAP DNA probe (A). Representative micrographs of three independent experiments. Scale bar = 100 µm.

 

Figure 4
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  		Figure 4 Immunolocalisation of PACAP within the mouse ovary. Very low signals for PACAP were detected in eCG-treated ovaries (A), whereas a strong immunoreactivity was observed in the GCs of antral follicles 6 h after hCG stimulation (B, arrow). Representative micrographs of three independent experiments. Scale bar = 100 µm.

 
In vitro stimulation of PACAP
Granulosa cells, isolated from preovulatory follicles obtained from eCG-treated mice, were incubated for 6 h in medium alone (C), or in the presence of 100 ng/ml follicle-stimulating hormone (FSH) or LH. At the end of the culture, cells were processed for RNA extraction and the presence of PACAP was assessed by semiquantitative RT-PCR. The expression levels of PACAP were compared with those attained in vivo by granulosa cells 6 h after hCG injection. After gonadotrophin stimulation, we observed a significant increase in PACAP mRNA, which reached levels that were slightly, though not significantly, lower than those obtained after in vivo stimulation (Fig. 5Go).


Figure 5
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  		Figure 5 In vitro stimulation of PACAP mRNA. GCs isolated from preovulatory follicles obtained from eCG-treated animals were incubated for 6 h in medium alone (C), or in the presence of 100 ng/ml FSH or LH. Expression levels were compared with those observed in GCs obtained from hCG-stimulated mice (hCG). Total RNA was extracted and analysed by semiquantitative RT-PCR for 30 cycles of amplification. ß-Actin was used to normalise mRNA levels. The figure is representative of three independent experiments carried out on separate animals.

 
Expression of PAC1 receptor
To characterise the expression of PAC1-R in mouse ovary, total RNA was extracted from whole ovaries, isolated granulosa cells and residual ovarian tissues, as described above. Using the primer pair common to all splice variants of the receptor, located at the 5'-end of the PAC1-R mRNA (PAC1R-Fw, PAC1R-Rv; see Fig. 1Go and Table 1Go), we detected two different products. In addition to the expected 317 bp fragment, we observed a fragment of 254 bp generated by the alternative splicing of the exon encoding a 21-aa domain in the N-terminal extracellular region of PAC1-R (Pantaloni et al. 1996; Fig. 6Go). The expression analysis of PAC1-R mRNA in whole ovaries shows that it is already present before gonadotrophin stimulation and is transiently stimulated between 3 and 6 h after hCG injection (Fig. 6AGo). When the GC population was analysed separately, we observed a similar pattern of expression, with an induction of PAC1-R-mRNA after hCG stimulation (Fig. 6BGo). In the residual ovarian tissues obtained from unstimulated animals, we observed a level of expression of PAC1-R mRNA similar to that observed in GCs but no modulation by gonadotrophin (data not shown).


Figure 6
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  		Figure 6 Effect of gonadotrophin stimulation on PAC1-R gene expression detected by multiplex RT-PCR. Ovaries were collected from untreated 22-day-old immature mice (22d), 48 h after eCG stimulation (0), or at different times after hCG treatment. Total RNA, extracted from whole ovaries (A) or GCs (B), was subjected to multiplex RT-PCR, as indicated in the Materials and Methods section, using PAC1R primers, and the ß-actin primer set 1 (Table 1Go) as the internal control. Aliquots of each PCR product were electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. The expression levels of the main PCR product (317 bp) were analysed by densitometric evaluation of the bands with a chemiluminescence detection system (raytest). PAC1-R values were normalised by their respective ß-actin values and are expressed as fold induction versus 22d arbitrarily set equal to 1. Values represent the mean ± S.E.M. of three independent experiments carried out on separate animals. The 254 bp product revealed the presence in the ovary of the very short isoform generated by the splicing of the 63 bp fragment at the N-terminal extracellular region of the PAC1-R gene (Pantaloni et al. 1996). *P<0.05; **P<0.01; ***P<0.001 versus 22d.

 
To test whether the aforementioned different splice variants were present in mouse preovulatory ovary, we performed RT-PCR on RNA extracted from whole ovaries 3 h after hCG injection (ov) with primers flanking the site of insertion of the hip/hop cassettes (hip/hop-Fw, hip/hop-Rv; Fig. 1Go and Table 1Go). This primer pair gave rise to two amplification products corresponding to the 181 bp PAC1-R short isoform, with neither hip nor hop exons, and a 265 bp with only one hip or hop cassette, or a mixture of the two isoforms (Fig. 7Go, lane a). Both isoforms were induced by hCG treatment (data not shown). Mouse brain, used as a positive control, expressed the same splice variants as those detected in ovary (Fig. 7Go, lane c).


Figure 7
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  		Figure 7 Analysis of PAC1-R isoforms in the mouse ovary. Total RNA from ovaries (ov), obtained 3 h after hCG stimulation, was subjected to RT-PCR for 50 cycles of amplification with the hip/hop and hop primers (Table 1Go and Fig. 1Go). Mouse brain (br) was used as a positive control. Aliquots of each PCR product and DNA molecular mass markers (Promega 100 bp DNA ladder) were electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. The hip/hop primer pair amplified two products corresponding to the short form at 181 bp, i.e. the one without either cassette, and the form with only one cassette, i.e. hip or hop, at 265 bp (lane a and c). The hip/hop Fw and hop Rv primer pair revealed the presence of one band of 148 bp, corresponding to the hop cassette (lanes b and d). The RT-PCR products obtained (control) were digested with PvuII, a restriction enzyme specific for the hop1 sequence. The presence of hop1 is shown by the digestion of the 265 bp band into two fragments of 148 and 117 bp, while the 181 bp band was not affected (hip/hop; lanes e and g), and by the digestion of the 148 bp band (hop) to 117 bp (lanes f and h). The incomplete digestion of the hop product at 264 and 148 bp indicates the presence of the hop2 isoform. The figure is representative of three independent experiments carried out on separate animals.

 
Since there is no mouse hip sequence in NCBI GenBank, in order to test the presence of the hip cassette in the mouse ovary, we based our analysis on the high homology (93%) of the mouse PAC1-R with the rat gene. In rat, the hip sequence is cleaved by the AvaI restriction enzyme. When we digested the amplified products obtained with the hip/hop primer pair, we did not observe any cleavage fragments (data not shown). Amplification with the hip/hop-Fw and hop-Rv (within the hop cassette, Fig. 1Go) primer pair resulted in only one product at 148 bp (Fig. 7Go, lanes b and d), confirming the presence of the hop1/hop2 isoforms and the absence of the hip isoform in both ovary and brain.

In order to distinguish between the hop1 and 2 isoforms, we exploited the nucleotide triplet insertion at the 5'-end of the hop1 exon, which generates a recognition site for the restriction enzyme PvuII. After complete digestion of the PCR products obtained with the hip/hop primers, the 181 bp band remained unchanged, while we observed digestion of the 265 bp band, accompanied by the appearance of two shorter fragments (Fig. 7Go, lanes e and g) of 117 and 148 bp, which thus confirmed the presence of the hop1 isoform. The incomplete digestion of the 265 bp band suggested the contemporary presence of the hop2 isoform. Similar conclusions were drawn when the product obtained with the hip/hop-Fw and hop-Rv primers was digested with PvuII. The 148 bp band was partially digested and a 117 bp band appeared (Fig. 7Go, lanes f and h).

Expression of VIP receptors
Analysis of VPAC1-R mRNA in whole ovary showed that the transcripts were present in untreated 22-day-old immature animals (22d) and were significantly down-regulated after gonadotrophin stimulation. VPAC1-R mRNA decreased after eCG stimulation and almost disappeared after hCG stimulation (Fig. 8Go). VPAC1-R was predominantly expressed in the residual ovarian tissue, in which it decreased markedly after gonadotrophin stimulation, whereas in GCs it was constantly present at very low levels (Fig. 9Go).


Figure 8
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  		Figure 8 The effect of gonadotrophin stimulation on VPAC1-R gene expression detected by multiplex RT-PCR on whole mouse ovaries. Total RNA was extracted from ovaries obtained from untreated 22-day-old immature mice (22d), 48 h after eCG stimulation (0), or at different times after hCG treatment, and subjected to multiplex RT-PCR, as indicated in the Materials and Methods section. An aliquot of each PCR product was electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. The ß-actin (primer set 2; Table 1Go) was used as an internal control. The expression levels were analysed by densitometric evaluation of the bands with a chemiluminescence detection system (raytest). VPAC1-R values were normalised by their respective ß-actin values and are expressed as percentage of inhibitionversus 22d arbitrarily set equal to 100. Values represent the mean ± S.E.M. of three independent experiments carried out on separate animals. *P<0.01 versus 22d.

 

Figure 9
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  		Figure 9 VPAC1-R expression in GCs and residual ovarian tissues. Total RNA, extracted from GCs and residual ovarian tissue (TI) obtained from ovaries of untreated 22-day-old immature mice (22d) and eCG-treated (eCG) mice was subjected to multiplex RT-PCR using ß-actin primer set 2 (Table 1Go) as an internal control. An aliquot of each PCR product was electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. In 22-day-old animals the receptor was predominantly expressed in TI; in GCs it was present in very low levels. After gonadotrophin stimulation, the expression levels decreased markedly in TI. The figure is representative of three independent experiments carried out on separate animals.

 
VPAC2-R expression slightly increased between 1 and 9 h following hCG stimulation in both whole ovary (Fig. 10Go) and the various cell populations considered separately (data not shown).


Figure 10
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  		Figure 10 The effect of gonadotrophin stimulation on VPAC2-R gene expression detected by multiplex RT-PCR on whole mouse ovaries. Total RNA was extracted from ovaries obtained as described in Fig. 2Go and subjected to multiplex RT-PCR, as indicated in the Materials and Methods section. An aliquot of each PCR product was electrophoresed onto 1.5% agarose gel and stained with ethidium bromide. The ß-actin (primer set 2; Table 1Go) was used as an internal control. The expression levels were analysed by densitometric evaluation of the bands with a chemiluminescence detection system (raytest). VPAC2-R values were normalised by their respective ß-actin values and are expressed as fold induction versus 22d arbitrarily set equal to 1. Values represent the mean ± S.E.M. of three independent experiments carried out on separate animals.

 
Evaluation of receptor activity
To determine the presence of functional receptors and ascertain which receptor is physiologically active in the preovulatory follicle, we studied the effects of PACAP and VIP on GC apoptosis. Follicles obtained from eCG-treated animals were cultured for 24 h in the absence of serum and in the presence of increasing concentrations of PACAP and VIP (from 10–9 to 10–7 M). At the end of the culture, GCs were mechanically isolated by puncturing the large antral follicles and were stained with TUNEL to assess the presence of apoptotic cells. GCs obtained from follicles immediately after isolation displayed very weak signs of apoptosis (2 ± 0.5%). In GCs obtained from follicles incubated in serum-free medium for 24 h, apoptosis increased to 61.2 ± 5.2%, with PACAP and VIP proving equally effective in preventing GC apoptosis (Fig. 11Go). To further determine the contribution of the different receptor subtypes to this inhibitory effect, follicles were incubated with 10–7 M PACAP or VIP in the presence of 5 x 10–7 M PACAP/VIP receptor antagonists. The antagonists used were: PACAP (6–38; P1), a PAC1-R and, to a lesser degree, VPAC2-R selective antagonist, (D-P-chloro-Phe6,Leu17)-VIP (V1), a moderately potent VPAC1-R antagonist and a hybrid of neurotensin (6–11), and VIP (7–28; P1/V2), a moderately potent PAC1-R antagonist and a weak VPAC2-R antagonist (Dickinson et al. 1997). All the receptor antagonists significantly reverted PACAP action, with P1 proving the most effective, though only V1 and P1/V2 inhibited VIP action, thereby supporting the presence of all three receptor subtypes in the follicle.


Figure 11
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  		Figure 11 Effect of PACAP and VIP on GC apoptosis. GCs were isolated from follicles obtained from eCG-treated mice and incubated in serum-free medium for 24 h alone (C), with 100 ng/ml LH, or increasing concentrations of PACAP and VIP (from 10–9 to 10–7 M). Values represent the mean ± S.E.M. of three to five independent experiments. *P<0.05, **P<0.01, ***P<0.001.

 
Western blot of VPAC1-R
Although VPAC1-R mRNA significantly decreased after gonadotrophin stimulation, the VPAC1-R antagonist nonetheless inhibited PACAP and VIP action on GC apoptosis (Fig. 12Go). Therefore, we investigated by western blot the presence of this receptor in whole ovary. As shown in Fig. 13Go, VPAC1-R was present in untreated 22-day-old immature animals and was still present at similar levels after eCG stimulation. The heterogeneity in molecular mass observed for VPAC1-R was probably due to differences in levels of glycosylation, as demonstrated in other human tissues (Bajo et al. 2000).


Figure 12
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  		Figure 12 Percentage of apoptosis in granulosa cells isolated from early antral follicles cultured for 24 h in the absence of serum (C) or supplemented with PACAP (10–7 M; upper panel) without (none) or with 10–6 M PAC1R antagonist PACAP (6–38; P1), 10–6 M VPAC1-R antagonist (V1) or PAC1/VPAC2-R antagonist (P1/V2); (lower panel) GCs isolated as shown in the upper panel but cultured in the presence of VIP (10–7 M) with or without receptor antagonists. Values represent the mean ± S.E.M. of three to six independent experiments. *P<0.05; **P<0.01.

 

Figure 13
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  		Figure 13 Western blot analysis of VPAC1-R in whole mouse ovary. Western blot analysis was performed using whole ovarian lysate from untreated 22-day-old immature mice (22d) and eCG-treated mice (eCG). The analysis shows the presence of three different bands in both samples. Tubulin was used as an internal control. Molecular mass standards were run in a parallel lane.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
We have previously demonstrated that both PACAP and VIP act on growing mouse follicles and that PACAP/VIP receptors are present in 14-day-old mice (Cecconi et al. 2004). In this study, we further characterised the presence of PACAP and their receptors in whole mouse ovary and in isolated GCs of immature mice, at different times after gonadotrophin stimulation.

Here, we demonstrate stimulation of PACAP mRNA in whole mouse ovaries after the LH surge, thus confirming the results by Park et al.(2003). In addition, we show that PACAP expression increases predominantly in GCs of preovulatory follicles up to 6 h after hCG injection, while PACAP mRNA levels in the residual ovarian tissue are significantly lower.

With regard to the PACAP/VIP receptors, we have previously shown, in ovaries from juvenile mice, that VPAC1-R is the most abundant receptor, that VPAC2-R levels are lower and that those of PAC1-R are very low (Cecconi et al. 2004). Here, we show that these three receptors are also present in the ovary of untreated 22-day-old immature animals. Upregulation by gonadotrophin stimulation is significant in PAC1-R in the GC compartment, and mild in VPAC2-R in both cell populations. By contrast, VPAC1-R is prevalently expressed in residual ovarian tissues, and is significantly downregulated by gonadotrophin. The localisation of VPAC1-R in this study is in keeping with that observed in rat ovary in which this receptor is observed above all in theca cells and in the proximity of blood vessels (Vaccari et al. 2006).

PAC1-R has been described to have many spliced variants arising from differences in the splicing of the PAC1-hnRNA. Knowing which isoforms are expressed in the different cell populations is of interest because these isoforms display differences in ligand affinity, coupling efficiency and activation of signal transduction pathways (Spengler et al. 1993, Pantaloni et al. 1996). We therefore investigated, in more detail, the presence of the different splice variants in mouse ovary. We found that the short and the hop1/hop2 variants are predominantly expressed in this organ, and that these variants do not appear to be modulated by hCG, while the hip isoform is not detectable in mouse ovary or brain.

This expression pattern is in agreement with results obtained in rat ovary by Gras et al.(2000), though the presence of the other PAC1-R isoforms has been shown by Scaldaferri et al.(1996). The presence of the short and the hop1/hop2 variants give GCs the ability to activate both AC and PLC (Spengler et al. 1993).

The finding that PACAP and VIP are equipotent in inhibiting GC apoptosis in follicles cultured in the absence of serum is consistent with the presence of functional PACAP and VIP receptors. These results were confirmed by the use of PACAP/VIP receptor antagonists. As expected, the RT-PCR data showed that PACAP (6–38) inhibited PACAP action on apoptosis, though not that of VIP, and that the VPAC2-R antagonist inhibited both PACAP and VIP. Unexpectedly, we also observed an inhibitory effect with the VPAC1-R antagonist despite very low VPAC1-R mRNA levels. However, western blot analysis revealed that the VPAC1-R protein was indeed still present, thus explaining the data obtained on GC apoptosis.

The transient, gonadotrophin-dependent production of PACAP and PAC1-R in the preovulatory follicles suggests that PACAP may play a significant role around the time of ovulation. Indeed, we have previously shown that rat oocyte meiotic maturation and the production of enzymes correlated with ovulation, such as tPA and uPA are dependent on PACAP stimulation (Apa et al. 2002); moreover, we have unpublished observations showing a beneficial effect of PACAP on mouse oocyte maturation and cumulus mucification (manuscript in preparation).

Although VIP immunoreactivity has been found to be located in interstitial tissue and to be associated with blood vessels and theca layers of follicles in several species (Ahmed et al. 1986, Kannisto et al. 1986, Jorgensen et al. 1991, Hulshof et al. 1994), and VIP mRNA has been detected in rat ovary (Gozes & Tsafriri 1986), we did not detect mRNA for VIP at any of the times considered. Therefore, our data are in agreement with the fact that this peptide has been shown to originate in extrinsic innervations. In fact, the lack of radioimmunoassayable VIP levels following the transection of the ovarian nerves indicates that ovarian VIP derives mostly from extrinsic innervation of the gland (Dees et al. 1986, Advis et al. 1989).

The presence of VPAC1-R and VPAC2-R in both mouse GCs and residual ovarian tissue combined with the fact that VIP has been shown to prevent GC apoptosis in rat and mouse ovary as efficiently as PACAP (Flaws et al. 1995, Cecconi et al. 2004) suggests that this peptide also plays a role in the preovulatory follicle. However, the different localisation of PACAP and VIP suggests that these two peptides play different, though synergistic, roles in the preovulatory follicle. It is noteworthy that VPAC2-Rs have been described in the smooth muscles of male and female reproductive tracts and blood vessels (Usdin et al. 1994, Vaccari et al. 2006), and that VIP has been detected mainly outside the follicles, where it exerts a relaxant effect on the ovarian arteries (Jorgensen 1991). We can therefore hypothesise that VIP contributes to the increase in blood flow around preovulatory follicles observed after the LH surge (Acosta et al. 2003). This increased ovarian stromal blood flow may, in turn, lead to a greater delivery of gonadotrophins to the granulosa cells of preovulatory follicles (Redmer & Reynolds 1996). The gonadotrophin would consequently stimulate the production of PACAP in the preovulatory follicle. The fact that PACAP induces genes related to ovulation and luteinisation, and mediates some of the effects of LH on granulosa cell differentiation at the time of ovulation (Gras et al. 1999, Lee et al. 1999, Park et al. 2000), suggests that PACAP may serve as an ovarian physiological mediator of gonadotrophins in the ovulatory process.

Further studies are warranted to evaluate the respective roles of PACAP and VIP in ovarian physiology.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The polyclonal rabbit anti-mouse antibody to PACAP was kindly provided by Professor Arimura of the Tulane University, Louisiana. Highly purified ovine LH (NIDDK-o-LH-26,BIO) and ovine FSH (NIDDK-o-FSH-19-SIAFF,BIO) were kindly provided by Dr Parlow (National Hormone and Pituitary Program of the NIH). We thank Mr S Greci for his excellent technical assistance and Mr Lewis Baker for reviewing the English in the manuscript. This work was supported by grants from the MIUR co-fin 2003 to R C and 2005 to M S, from ‘La Sapienza’ University of Rome 2004 to R C and from the Department of Health Special Program 2004 to M S. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 31 January 2007
First decision 27 March 2007
Accepted 30 April 2007


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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