| HOME | HELP | CONTACT US | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, Via A. Scarpa, 14, 00161 Rome, Italy
1 Department of Experimental Medicine, University of L’Aquila, Rome, Italy
(Requests for offprints should be addressed to R Canipari; Email: rita.canipari{at}uniromal.it)
* (S Vaccari and S Latini contributed equally to this work) 
(S Vaccari is now at Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Drive Stanford, CA 94305-5317, USA)
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PACAPs, VIP, and their receptors are expressed 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,b), which suggests that they may not play a neuroendocrine role alone. Interestingly, PACAP stimulates various ovarian functions, including steroidogenesis, cAMP accumulation, and plasminogen activator (PA) production in rat granulosa cells (Zhong & Kasson 1994, Heindel et al. 1996, Apa et al. 2002), accelerates meiotic maturation in cumulus-enclosed rat oocytes (Apa et al. 1997), and inhibits apoptosis in preovulatory follicles (Lee et al. 1999), thus indicating that it plays an important role in the female reproductive system. We have also shown a direct action of PACAP on denuded oocytes (Apa et al. 1997).
Here, we describe the characterization and the signal transduction pathway of the three PACAP/VIP receptors in the various cellular compartments of the ovarian follicle. We reveal that PACAP receptors are present on the surface of denuded germinal vesicle (GV) oocytes, thus demonstrating a direct action of the peptide on these cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The materials used in the present study were obtained from the following sources: equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) from Intervet (Livorno, Italy); [125I]cAMP and [3H]inositol-1.4.5-trisphosphate from NEN, Perkin Elmer Life Science (Monza, Italy); PACAP and VIP from Calbiochem (San Diego, CA, USA); PACAP/VIP receptor antagonists PACAP 6–38 (H-2734), and a hybrid of neurotensin (6–11) and VIP (7–28) (H-9935) from Bachem (Bubendorf, Swisse); Mebstain Apoptosis Kit Direct from MBL International (Woburn, MA, USA); the antibodies to VPAC1 H-130 (sc-30019) and VPAC2 H-50 (sc-30020) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, USA); Dulbecco’s modified Eagle medium (DMEM), minimum essential medium with Earl’s salts (MEM), Hank’s balanced salt solution and fetal calf serum (FCS) from Gibco (Grand Island, NY, USA); dibutyryl cAMP (dbcAMP), cAMP, VIP receptor antagonist (D-P-Chloro-Phe6, Leu17)-VIP, and all other reagents from Sigma. Highly purified ovine follicle-stimulating hormone (NIDDK-o-FSH-19-SIAFP, BIO) was kindly provided by Dr Parlow (National Hormone and Pituitary Program of the NIH).
Animals
Immature female Wistar rats were purchased from Charles River (Como, Italy). They were housed in groups, maintained in controlled temperature (25 °C) and light (12 h light/day) conditions, and given a regular supply of food and water and allowed to feed ad libitum. Animals were maintained in accordance with the NIH Guide for Care and Use of Laboratory Animals. Experimental protocols were approved by the University ‘La Sapienza’ Committee for Animal Care and Use. Animals aged from 3 to 25 days were killed by cervical dislocation and the ovaries collected for further analysis. Twenty-five-day-old rats were either killed (T0, untreated rats) or injected subcutaneously with 10 IU eCG. After 48 h, the latter group was either killed (eCG-rats) or injected with 10 IU hCG (hCG-rats) and killed after 6 h by cervical dislocation.
Granulosa and theca/interstitial cell cultures
Granulosa cell cultures were prepared from eCG-treated rats as previously described (Canipari & Strickland 1985). Briefly, the largest follicles from each ovary were punctured with a 25-gauge needle under stereomicroscope and gently pressed to release the granulosa cells. These cells were collected and cultured at a density 1.5 x 105/200 µl in MEM supplemented with 0.1% BSA, 2 mM glutamine and antibiotics (100 mM penicillin and 100 µg/ml streptomycin). Viability was estimated by the trypan blue dye exclusion method.
According to the procedure of Hwang et al.(1996), with minor modifications, theca/interstitial (TI) cells were obtained by digestion of the residual ovarian tissues from untreated rats, after granulosa cell isolation. Briefly, the residuals of the largest follicles were cut into small pieces, washed, and digested with collagenase in a two-step procedure. To obtain TI cells, the tissue was first digested in DMEM containing collagenase (1 mg/ml) and DNase (0.5 µg/ml) at 37 °C for 30 min in order to eliminate the adhering granulosa cells, then washed and digested in DMEM containing 4 mg/ml collagenase and 0.5 µg/ml DNase for 45 min at 37 °C. The cells were plated in growth medium (DMEM supplemented with 10% FCS, 5 mM glutamine, and antibiotics) at a density 2 x 105 cells in 6 cm Petri dishes and incubated until use. Viability was estimated by the trypan blue dye exclusion method. All incubations were carried out at 37 °C in a 5% CO2 atmosphere.
Early antral follicles between 320 and 400 µm diameter were mechanically dissected from ovaries of 19-day-old rats as previously described (Cecconi et al. 2004). Groups of 25 follicles were incubated on stainless steel grids in 
MEM supplemented with 0.3% BSA in the absence or presence of increasing concentrations of PACAP or FSH (100 ng/ml) for 24 h at 37 °C. In additional experiments follicles were incubated with PACAP and VIP in the presence of 10–6 M PACAP and VIP antagonists.
RNA extraction and RT-PCR
After the animals had been killed, ovaries were removed aseptically, freed from adherent tissues and stored, ready for RNA extraction, at –80 °C.
Total RNA from whole ovaries, granulosa cells, and TI cells was isolated by the single-step acid guanidinium thiocyanate–phenol–chloroform method (Chomczynski & Sacchi 1987). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis. Total RNA (1–2 µg) was reverse transcribed, in a final volume of 20 µl, using the SuperScript II kit (Gibco) according to the manufacturer’s instructions. The PCRs were carried out using Taq DNA polymerase (Roche) according to the manufacturer’s instructions.
For each primer set, the number of cycles for the PCR was chosen in the exponential phase of amplification, using the annealing temperature provided. For each sample, 10 µl PCR product was submitted to electrophoresis on agarose gel (1.5%) and stained with ethidium bromide. Amplified products were analysed using AIDA software (Advanced Image Data Analyzer, 2.11) and mRNA levels normalized against the expression of ribosomal protein s16 mRNA.
Controls for DNA contamination were performed with gene-specific primers on RNA without reverse transcriptase treatment.
Theprimers used to amplify PAC1-R, VPAC1-R, VPAC2-R, the ribosomal protein S16 (S16), and the follicle-stimulating hormone receptor (FSH-R) are shown in Table 1
. Primers for PAC1-R were chosen in a region that allowed the detection of all splice variants.
|
According to the method of Fiorenza & Mangia (1998), mRNA from rat oocytes was amplified by PCR with slight modifications. Briefly, GV oocytes, obtained by puncturing ovarian follicles of eCG-treated rats, were mechanically isolated from the surrounding cumulus cells and either immediately lysed for RNA extraction or allowed to mature for 6 h in DMEM supplemented with 0.23 mM sodium pyruvate and 5% FCS (Met I-oocytes), then treated for RNA isolation. Ovulated metaphase II-oocytes (Met II) were obtained from the oviducts of eCG–hCG-primed animals, 15 h after hCG injection. The oocytes were freed from cumulus cells (CCs) by hyaluronidase treatment.
In order to remove the zona pellucida (ZP), the oocytes were incubated at room temperature for 1–2 min in protein-free Hepes-buffered medium M2 (Quinn et al. 1982) containing 0.5% Pronase E (Sigma) (Canipari et al. 1988). Groups of five oocytes were transferred to a 0.5 ml tube containing 2 µl H2O supplemented with 1 U/µl RNasin Ribonuclease Inhibitor (Promega). Tubes were rapidly frozen on dry ice and stored at –80 °C until used. Immediately before the assay, the oocytes were diluted by adding 2 µl diethylpyrocarbonate (DEPC)-treated water. Total RNA was extracted by rapidly thawing and freezing the tube twice. The lysate was then digested with 40 U/ml RNase-free bovine pancreatic DNase I at 37 °C for 7 min to eliminate the genomic DNA, then heated at 65 °C for 5 min to inactivate the DNase I and denature the RNA. Total RNA was reverse transcribed using the Sensiscript Reverse Transcriptase Kit (Qiagen S.p.a., GnbH, Germany), according to the manufacturer’s instructions. Controls were performed by omitting the Sensiscript enzyme during RT. The PCR step was performed by adding 15 pmole of each specific primer and 2.5 U Taq DNA polymerase (Roche) in a final volume of 60 µl. The primers used to amplify PAC1-R and S16 are shown in Table 2. VPAC1-R and VPAC2-R were the same as those used for the granulosa and TI cells (Table 1
).
|
Cytoplasmic Ca2+ concentration ([Ca2+ ]i) was measured by dual wavelength fluorescence of single cells loaded with the Ca2+ -sensitive intracellular indicator fura-2-acetoxymethylester (AM), as described by Paniccia et al.(1995), with slight modifications.
Zona-free oocytes, 5–10, washed and transferred to M2 (without BSA) on glass coverslips that had been precoated with concanavalin A (Con A, 0.2 mg/ml in PBS), forming the base of thechamberfor theCa2+ measurement(McGuinness et al. 1996). Oocytes were then loaded with 3 µM fura-2/AM in serum-free, but otherwise complete, MEM at 37 °C in 5% CO2 for 60 min. Coverslips were then rinsed twice with Krebs–Henseleit–Hepes buffer (KHH: 140.7 mM Na+ , 5.3 mM K+ , 132.4 mM Cl–, 0.98 mM PO4 2–, 1.25 mM Ca2+ , 0.81 mM Mg2+ , 20.3 mM HEPES, and 5.5 mM glucose). [Ca2+ ]i-dependent fluorescence was measured by means of an AR-CM microfluorimeter (Spex Industries, Edison, NJ, USA) connected with a Diaphot TMD inverted microscope equipped with a CF x 40 fluor objective (Nikon Corp., Tokyo, Japan). Recordings were performed at 340 and 380 nm excitation wavelengths. Emission, collected by a photomultiplier carrying a 510 nm cut-off filter, from 340 to 380 nm and a real-time 340:380 nm ratio was recorded by an ASEM Desk 2010 computer (ASEM S.p.A., Buia, Italy). Calibration of the signal was obtained at the end of each experiment by maximally increasing intracellular Ca2+ -dependent fura-2 fluorescence with 5 µM Ca2+ -ionophore ionomycin, followed by the recording of minimal fluorescence after the addition of 7.5 mM EGTA and 60 mM Tris–HCl, pH 10.5 [Ca2+ ]i, was calculated as previously described (Grynkiewicz et al. 1985).
Assay of cAMP
Cultured TI cells were incubated for 1 h in serum-free medium and then for 2 h in the presence of various hormones. At the end of the incubation period, the samples were processed as previously described (Apa et al. 1997), with slight modifications. Stimulation was stopped by means of ice-cold 10% trichloroacetic acid (TCA), and the cells were scraped and sonicated twice for 5 s at 5 W. The lysate was centrifuged and the supernatant extracted thrice with two volumes of water-saturated diethyl ether. The amount of cAMP was measured by RIA (Steiner et al. 1972). Samples were acetylated before the assay, according to the procedure of Harper & Brooker (1975). The RIA had a sensitivity of 2–4 fmol cAMP, an intraassay coefficient of variation of 5%, and an interassay coefficient of variation of 10.3%. The standard curve was calculated using a log-linear curve fit with %B/B0 (y-axis) against cAMP. The values were normalized to the milligrams of proteins present in the sample. Protein content was measured by the method of Lowry et al.(1951) using BSA as standard.
Phosphoinositide turnover
In order to measure phosphoinositide degradation, TI and granulosa cells were incubated for 48 h with 4 µCi/ml myo-[2-H3]inositol in DMEM supplemented with 5% FBS. After labeling, cells were rinsed thrice with Hank’s balanced salt solution and preincubated for 20 min with 20 mM LiCl. PACAP and VIP treatment were started a few minutes after LiCl addition, and stopped 30 min later by rapidly placing the culture plates on ice and replacing the medium with ice-cold 10% TCA. TCA was cleared away by extraction with 1 volume trichlorotrifluoroethane and trioctilamine (3:1). The samples were separated by ion exchange chromatography on Dowex 1 x 8–200 (Sigma) and eluted with ammonium phormiate solution at different concentrations. Samples were counted using a ß-counter (Beckman Coulter, Inc., NY, USA).
Immunofluorescence
Frozen sections (7 µm) obtained from ovaries of 23-day-old rats were mounted on Polysine-TM slides (Menzel-Glaser, Braunschweig, Germany), fixed in 4% formaldehyde, treated with 10% normal goat serum to minimize non-specific binding, and incubated for 20 h at 4 °C with antibodies to VPAC1-R (1:200) or VPAC2-R (1:200). The sections were extensively washed with PBS and incubated for 1 h at room temperature with a goat anti-rabbit IgG (Molecular Probes; 1:1000). As a negative control, the primary antibody was omitted and substituted with rabbit immunoglobulin G. Samples were analyzed using a Zeiss Axioplan fluorescence microscope (Carl Zeiss SpA, Milano, Italy).
Morphological analysis of granulosa cell apoptosis
Granulosa cell apoptosis was evaluated as previously described (Cecconi et al. 2004). Briefly, early antral follicles were mechanically dissected from 19-day-old untreated rats and cultured as described previously. Granulosa cells were released in the medium immediately before or after 24 h of follicle culture. Cells from single follicles were fixed for 15 min in 4% paraformaldeyhde/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 labeling) 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.
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 or paired Student’s t-test for comparison of data derived from two groups. Values with P < 0.05 were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In order to determine the expression of different PACAP receptors in rat ovary, total RNA was extracted from whole ovaries obtained from untreated, eCG-, and hCG-treated rats. Semi-quantitative RT-PCR (25 cycles of amplification) showed the presence of PAC1-R in untreated and hCG-treated rats and very low levels in eCG-treated rats, as previously demonstrated (Park et al. 2000), but no signal for the presence of the other two receptors (Fig. 1). Positive signals for VPAC1-R and VPAC2-R were observed only after increasing cycles of amplification, thus suggesting a lower level of expression for these two genes. VPAC2-R was evidenced after 30 cycles and apparently not modulated by gonadotropin treatment (Fig. 1). VPAC1-R was seen only after 35 cycles and decreased slightly after gonadotropin stimulation (Fig. 1).
|
). This receptor was not observed in TI cells also at 35 cycles of amplification (data not shown). Positive signals for VIP receptors were observed after further amplification confirming lower levels of expression. VPAC1-R was observed only in TI cells, and VPAC2-R was observed in bothgranulosa and TI cells; VPAC2-R was slightly stimulated by hCG in TI cells (Fig. 2).
|
). A faint band was also present in TI cells obtained by means of mechanical dissociation of the follicles and was presumably due to granulosa cell contamination. FSH-R mRNA was not expressed in in vitro cultured TI cells, suggesting the absence or very low contamination by granulosa cells (Fig. 3). As already observed in ex vivo TI cell preparations after 35 cycles of amplification, a positive signal for VPAC1-R and VPAC2-R was identified in cultured TI cells, although none was present for PAC1-R. VPAC2-R was the predominant form in TI cells (Fig. 4).
|
|
, all the receptors were expressed in 3-day-old rats although higher numbers of cycles of amplification were needed to detect VIP receptors suggesting lower level of expression for these two genes also in immature ovaries. PAC1-R markedly increased after the 12th day of age (Fig. 5A), VPAC1-R significantly decreased with increasing age (Fig. 5B), while VPAC2-R remained constant through time (Fig. 5C).
|
To localize the VIP receptors, immunofluorescence analysis was performed on ovarian sections obtained from 23-day-old rats. The results showed that VPAC1-R was predominantly found in association with the blood vessel wall and in the stroma near follicles (Fig. 6). We observed a strong immunoreactivity at the level of the ovarian hilus (Fig. 6A), where the ovarian arteries enter the ovary (Hossain & O’Shea 1983). Consistent with mRNA expression pattern, VPAC2-R was detected ubiquitously in the ovary (Fig. 7) and, as already shown (Bajo et al. 2000), a positive signal was observed in the oviduct (Fig. 7A).
|
|
As previously shown (Apa et al. 2002), PACAP was able to act directly on denuded oocytes, delaying meiotic maturation by modulating their intracellular cAMP levels. Therefore, we investigated oocytes for the expression of PACAP receptors. In order to eliminate any possible contamination by granulosa cell remnants, the ZP was first removed from denuded oocytes by pronase treatment (Canipari et al. 1988), then RNA extraction and RT-PCR were performed as described in the Materials and Methods. A positive signal for the expression of PAC1-R was detected in GV oocytes after 40 cycles followed by a second amplification for 30 cycles on an aliquot of the first amplification. No signal was detected for VPAC1-R and VPAC2-R under the same conditions (Fig. 8). No signal for PAC1-R was observed in in vivo matured Met I- and Met II-oocytes (data not shown).
|
). No response was detected, in similar conditions, in Met I- and Met II-oocytes (data not shown). This result indicates that functional PAC1-Rs are only present on the oocyte surface at the GV stage.
|
In order to determine the signal transduction pathway associated with the activation of PACAP and VIP receptors present in granulosa and TI cells, we studied the effect of the two peptides on inositol monophosphate (IP1), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) production. Granulosa cells were cultured for 48 h in the presence of myo-[2-H3]inositol, pretreated for 10 min with LiCl, then stimulated with increasing concentrations of PACAP and VIP. Gonadotropin-releasing hormone (GnRH) was used as a positive control for IP production of granulosa cells (Anderson et al. 1996). Figure 10 shows that both PACAP-38 and PACAP-27 activate PLC, which in turn, catalyses the breakdown of polyphosphoinositides. Treatment with VIP did not significantly affect IP production in these cells, even at high concentrations (10–6 M). When the same experiment was performed on TI cells, cultured for 48 h in the presence of PACAP and VIP, no effect was detected on IP production (data not shown).
|
).
|
In order to evaluate the presence of functional receptors in granulosa or TI cells, we studied the effect of PACAP and VIP on granulosa cell apoptosis. Cells, obtained from 22-day untreated rats, were cultured either isolated or in whole follicles. The presence of apoptotic cells was evaluated in granulosa cells after 24 h of culture in the presence of 100 ng/ml FSH, as a positive control, or increasing concentrations of PACAP (from 10–9 to 10–7 M). To this end granulosa cells were stained with Hoechst 33258 or with TUNEL at the end of culture. Granulosa cells obtained from early antral follicles immediately after isolation did not show detectable signs of apoptosis (4 ± 0.8%). In granulosa cells obtained from follicles incubated in serum-free medium for 24 h, apoptosis increased to 22.3 ± 1.5% and PACAP inhibited apoptosis in a dose-dependent manner (24 ± 1.5, 17 ± 1.2, 12 ± 0.9%, 10–9, 10–8, and 10–7 respectively). Levels of apoptosis similar to those obtained with FSH (14 ± 1.1%) were obtained with 10–7M PACAP. As already shown (Flaws et al. 1995) also VIP inhibited granulosa cell apoptosis when cultured in whole follicles (Fig. 12B) suggesting the presence of VIP receptors in the whole follicles.
|
To further characterize the contribution of the different receptor subtypes to this inhibitory effect, follicles were incubated with PACAP (Fig. 12A) and VIP (Fig. 12B) in the presence of PACAP/VIP receptor antagonists. The antagonists utilized were: PACAP (6–38) a PAC1-R, and to a lesser degree VPAC2-R selective antagonist (D-P-Chloro-Phe6, Leu17), VIP, a moderately potent VPAC1-R antagonist and a hybrid of neurotensin (6–11) and VIP (7–28), a moderately potent PAC1-R antagonist and a weak antagonist at VPAC2-R (Dickinson et al. 1997). As shown in Fig. 12A, all receptor antagonists significantly reversed PACAP inhibition of apoptosis, while PACAP (6–38) did not inhibit VIP action (Fig. 12B) supporting the presence of all three receptor subtypes in the follicle and the presence of PAC1-R predominantly in the GC compartment.
To determine if PAC1-R was effectively the predominant form present in granulosa cells, GCs isolated from early antral follicles, were incubated in the same conditions as described for whole follicles. PACAP and FSH prevented apoptosis (Fig. 12C), while VIP had no effect (data not shown). PACAP action was reversed by the addition of PACAP (6–38) and VIP (7–28), while (D-P-Chloro-Phe6, Leu17) VIP had no effect (Fig. 12C). These data are in accordance with the presence of PAC1-R and the absence of VPAC1-R in GCs. However, we cannot discriminate between the presence of PAC1-R and VPAC2-R due to the low selectivity of VIP (7–28).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We used the RT-PCR approach to determine the expression of PACAP/VIP receptors in the whole ovary. All three forms were found at all ages examined, though in different amounts. We found that the predominant form expressed in the rat ovary is PAC1-R, while VPAC1-R and VPAC2-R are less abundant.
In the preovulatory follicles, we analyzed granulosa cells, oocytes, and TI cells separately for the presence of VIP/ PACAP receptors. In accordance with the data published by Gras et al.(2000) in isolated granulosa cells, we observed abundant mRNA transcripts for PAC1-R, lower levels of mRNA for VPAC2-R and no mRNA for VPAC1-R. PAC1-R was the only receptor found to be statistically modulated by gonadotropin.
As far as TI cells are concerned, indeed, we found that VPAC2-R and VPAC1-R were both expressed in the TI compartment, whereas PAC1-R was not. This is in agreement with our previous demonstration that PACAP and VIP are equally effective in stimulating granulosa cells cultured within the whole follicle (Apa et al. 1997, 2002), which thus suggests the presence of VIP receptors in TI cells.
In agreement with functional (Apa et al. 1997) and binding data (Gras et al. 2000) reported in the literature, the present study provides direct evidence of the presence of PAC1-R and the absence of VIP receptors on GV oocytes. After having previously shown that PACAP, but not VIP, increases intracellular cAMP levels in denuded GV oocytes (Apa et al. 1997), we now demonstrate that 10–6 M PACAP stimulates Ca+ + mobilization in the same oocytes. These data are consistent with the presence of a functional PAC1 receptor in fully grown GVoocytes. PACAP receptors are already present in primordial germ cells in fetal gonads; moreover, PACAP has been shown to stimulate primordial germ cell proliferation via cAMP production (Pesce et al. 1996) and inhibit meiotic resumption in fully grown oocytes (Apa et al. 1997). However, after resumption of meiotic maturation, we observed a decrease in the levels of mRNA for these receptors, followed by a decrease in the number of functional receptors on the cell surface, as measured by an inability to stimulate Ca+ + mobilization. Therefore, at the time of PACAP production in preovulatory follicles, 3–6 h after the LH surge (Gras et al. 1996, Koh et al. 2000), this peptide is no longer able to directly interfere with oocyte maturation.
The presence and the distribution of functional PACAP/VIP receptors in granulosa and TI cells have been supported by metabolic studies. In isolated granulosa cells, PACAP stimulated both cAMP production (Apa et al. 1997) and phosphoinositide (IP) turnover (this paper), while VIP stimulated cAMP production alone at high concentrations in the presence of a phosphodiesterase inhibitor (IBMX) (Apa et al. 1997). These data demonstrate the presence of functional PAC1-R, the only one associated with the activation of both adenylate cyclase and PLC and of low number of VIP receptors. Conversely, in TI cells both PACAP and VIP stimulated cAMP but not IP breakdown, which is consistent with the presence of VIP receptors and the absence of PAC1-R.
Furthermore, here we demonstrate that PACAP and VIP both were able to prevent granulosa cell apoptosis in serum-free medium cultured follicles as already shown (Flaws et al. 1995, Lee et al. 1999), while only PACAP efficiently prevented apoptosis in isolated granulosa cells. These results together with the data obtained with the receptor antagonists further support the presence of functional VIP receptors on TI cells.
However, since TI cells are a very heterogeneous population we characterized VIP receptor localization in this compartment by immunofluorescence. In accordance with RT-PCR data, VPAC2-R was ubiquitously found in the ovary, while VPAC1-R was found in the proximity of follicles, but not in the GCs. The localization of VPAC1-R suggests an association with the smooth muscle cells located in the theca externa (Ko et al. 2006). A strong signal was found also in the blood vessel wall.
VIP has been detected in neonatal rat ovaries as early as 2 days after birth; VIP-containing nerve fibres are present in rat ovaries around blood vessels, as well as around follicles at different stages of development, in close proximity of the theca cell layers and occasionally between primordial follicles (Ahmed et al. 1986). The contemporary presence of VIP and its receptors (this paper) around preantral and antral follicles suggests a possible role of VIP in both growing and preovulatory follicles. Indeed, VIP has been shown to suppress granulosa cell apoptosis, to promote follicle survival in in vitro growing preantral rat follicles (Flaws et al. 1995), and to stimulate ovarian steroidogenesis (Davoren & Hsueh 1985, Ahmed et al. 1986). Moreover, it has been shown that it exerts a relaxant effect on the rabbit ovarian artery (Jorgensen 1991). Therefore, the localization of VIP and its receptors in association with blood vessels suggests that this neuropeptide might be involved in the regulation of ovarian blood flow. The increased ovarian stromal blood flow may lead to a greater delivery of gonadotropins to the granulosa cells of the developing follicles (Redmer & Reynolds 1996).
In conclusion, the different pattern of distribution of PAC1-R, VPAC1-R and VPAC2-R suggests distinct functional roles. The presence of VIP receptors around blood vessels, suggests an important role for this peptide in the blood flow regulation. In fact, perifollicular vascular expansion associated with increased rates of blood flow are developmentally important for the generation of a normal follicle and competent oocyte (Van Blerkom 2000).
The temporal and spatial distribution of PACAP, after the luteinizing hormone (LH) surge, suggests that the action of PACAP via PAC1-R may be restricted to a specific developmental window. The fact that PACAP has been shown to induce genes related to ovulation and luteinization, and to mediate 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 is involved in the preovulatory follicle and that it may serve as an ovarian physiological mediator of gonadotropin at the time of the ovulatory process. However, the presence of PACAP around growing follicles in cyclic rats, points to an additional role of this peptide in the regulation of growing follicle development at least in the adult ovary after the first LH surge. Indeed, we have recently demonstrated an inhibitory effect of PACAP on mouse preantral follicle growth and differentiation (Cecconi et al. 2004).
Indeed, activation of follicle development is under the influence of both stimulatory and inhibitory regulation (Skinner 2005). Abnormal regulation of primordial follicle development can affect the reproductive capacity of the female and menopausal onset, as shown in anti-Mullerian hormone null females (Durlinger et al. 1999). Further studies are needed to evaluate the role of PACAP and VIP in ovarian physiology.
|
Acknowledgements |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anderson L, Hillier SG, Eidne KA & Miro F 1996 GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells. Journal of Endocrinology 149 449–456.[Abstract]
Apa R, Lanzone A, Mastrandrea M, Miceli F, Macchione E, Fulghesu AM, Caruso A & Canipari R 1997 Effect of pituitary adenylate cyclase-activating peptide on meiotic maturation in follicle-enclosed, cumulus-enclosed, and denuded rat oocytes. Biology of Reproduction 57 1074–1079.[Abstract]
Apa R, Lanzone A, Miceli F, Vaccari S, Macchione E, Stefanini M & Canipari R 2002 Pituitary adenylate cyclase-activating polypeptide modulates plasminogen activator expression in rat granulosa cell. Biology of Reproduction 66 830–835.
Arimura A 1992a Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regulatory Peptides 37 287–302.[ISI][Medline]
Arimura A 1992b Receptors for pituitary adenylate cyclase-activating polypeptide. TEM 3 288–294.
Bajo AM, Juarranz MG, Valenzuela P, Martinez P, Prieto JC & Guijarro LG 2000 Expression of vasoactive intestinal peptide (VIP) receptors in human uterus. Peptides 21 1383–1388.[CrossRef][ISI][Medline]
Canipari R & Strickland S 1985 Plasminogen activator in the rat ovary. Journal of Biological Chemistry 260 5121–5125.
Canipari R, Bevilacqua A, Colonna R, De Felici M & Mangia F 1988 Actin synthesis is not regulated by granulosa cells in mouse growing and preovulatory oocytes. Gamete Research 20 115–124.[CrossRef][ISI][Medline]
Cecconi S, Rossi G, Barberi M, Scaldaferri L & Canipari R 2004 Effect of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on mouse preantral follicle development in vitro. Endocrinology 145 2071–2079.
Chomczynski P & Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytical Biochemistry 162 156–159.[ISI][Medline]
Davoren JB & Hsueh AJW 1985 Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. Biology of Reproduction 33 37–52.[Abstract]
Dickinson T, Fleetwood-Walker SM, Mitchell R & Lutz EM 1997 Evidence for roles of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) receptors in modulating the responses of rat dorsal horn neurons to sensory inputs. Neuropeptides 31 175–185.[CrossRef][ISI][Medline]
Durlinger AL, Kramer P, Karels B, de Jong FH, Uilenbroek JT, Grootegoed JA & Themmen AP 1999 Control of primordial follicle recruitment by anti-Mullerian hormone in the mouse ovary. Endocrinology 140 5789–5796.
Fiorenza MT & Mangia F 1998 Quantitative RT-PCR amplification of RNA in single mouse oocytes and preimplantation embryos. Biotechniques 24 618–623.[ISI][Medline]
Flaws JA, DeSanti A, Tilly KI, Javid RO, Kugu K, Johnson AL, Hirshfield AN & Tilly JL 1995 Vasoactive intestinal peptide-mediated suppression of apoptosis in the ovary: potential mechanisms of action and evidence of a conserved antiatretogenic role through evolution. Endocrinology 136 4351–4359.[Abstract]
Gottschall PE, Tatsuno I, Miyata A & Arimura A 1990 Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide. Endocrinology 127 272–277.[Abstract]
Gras S, Hannibal J, Georg B & Fahrenkrug J 1996 Transient periovulatory expression of pituitary adenylate cyclase activating peptide in rat ovarian cells. Endocrinology 137 4779–4785.[Abstract]
Gras S, Hannibal J & Fahrenkrug J 1999 Pituitary adenylate cyclase-activating polypeptide is an auto/paracrine stimulator of acute progesterone accumulation and subsequent luteinization in cultured periovulatory granulosa/lutein cells. Endocrinology 140 2199–2205.
Gras S, Hedetoft C, Pedersen SH & Fahrenkrug J 2000 Pituitary adenylate cyclase-activating peptide stimulates acute progesterone production in rat granulosa/lutein cells via two receptor subtypes. Biology of Reproduction 63 206–212.
Grynkiewicz G, Poenie M & Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260 3440–3450.
Harper JF & Brooker G 1975 Femtomole sensitive radioimmunoassay for cAMP and cGMP after 2'o-acetylation by acetic anhydride in aqueous solution. Journal of Cyclic Nucleotide Research 1 207–218.[ISI][Medline]
Heindel JJ, Sneeden J, Powell CJ, Davis B & Culler MD 1996 A novel hypothalamic peptide, pituitary adenylate cyclase-activating peptide, regulates the function of rat granulosa cells in vitro. Biology of Reproduction 54 523–530.[Abstract]
Hossain MI & O’Shea JD 1983 The vascular anatomy of the ovary and the relative contribution of the ovarian and uterine arteries to the blood supply of the ovary in the guinea-pig. Journal of Anatomy 137 457–466.
Hwang JJ, Lin SW, Teng CH, Ke FC & Lee MT 1996 Relaxin modulates the ovulatory process and increases secretion of different gelatinases from granulosa and theca-interstitial cells in rats. Biology of Reproduction 55 1276–1283.[Abstract]
Jorgensen JC 1991 Interaction between norepinephrine, NPYand VIP in the ovarian artery. Peptides 12 831–837.[CrossRef][ISI][Medline]
Kimura C, Ohkubo S, Ogi K, Hosoya M, Itoh Y, Onda H, Miyata A, Jiang L, Dahl RR & Stibbs HH 1990 A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs. Biochemical and Biophysical Research Communications 166 81–89.[CrossRef][ISI][Medline]
Ko C & Park-Sarge OK 2000 Progesterone receptor activation mediates LH-induced type-I pituitary adenylate cyclase activating polypeptide receptor (PAC(1)) gene expression in rat granulosa cells. Biochemical and Biophysical Research Communications 277 270–279.[CrossRef][ISI][Medline]
Ko C, Gieske MC, Al Alem L, Hahn Y, Su W, Gong MC, Iglarz M & Koo Y 2006 Endothelin-2 in ovarian follicle rupture. Endocrinology 147 1770–1779.
Koh PO, Kwak SD, Kang SS, Cho GJ, Chun SY, Kwon HB & Choi WS 2000 Expression of pituitary adenylate cyclase activating polypeptide (PACAP) and PACAP type I A receptor mRNAs in granulosa cells of preovulatory follicles of the rat ovary. Molecular Reproduction and Development 55 379–386.[CrossRef][ISI][Medline]
Lee J, Park HJ, Choi HS, Kwon HB, Arimura A, Lee BJ, Choi WS & Chun SY 1999 Gonadotropin stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP) messenger ribonucleic acid in the rat ovary and the role of PACAP as a follicle survival factor. Endocrinology 140 818–826.
Lowry OH, Rosebrough NJ, Farr AL & Randall RJ 1951 Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193 265–275.
Lutz EM, Sheward WJ, West KM, Morrow JA, Fink G & Harmar AJ 1993 The VIP2 receptor: molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide. FEBS Letters 334 3–8.[CrossRef][ISI][Medline]
McGuinness OM, Moreton RB, Johnson MH & Berridge MJ 1996 A direct measurement of increased divalent cation influx in fertilised mouse oocytes. Development 122 2199–2206.[Abstract]
Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, Culler MD & Coy DH 1989 Isolation of a novel 38 residue-hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. Biochemical and Biophysical Research Communications 164 567–574.[CrossRef][ISI][Medline]
Paniccia R, Riccioni T, Zani BM, Zigrino P, Scotlandi K & Teti A 1995 Calcitonin down-regulates immediate cell signals induced in human osteoclast-like cells by the bone sialoprotein-IIA fragment through a postintegrin receptor mechanism. Endocrinology 136 1177–1186.[Abstract]
Park HJ, Lee J, Wang L, Park JH, Kwon HB, Arimura A & Chun SY 2000 Stage-specific expression of pituitary adenylate cyclase-activating poly-peptide type I receptor messenger ribonucleic acid during ovarian follicle development in the rat. Endocrinology 141 702–709.
Pesce M, Canipari R, Ferri JL, Siracusa G & De Felici M 1996 Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates adenylate cyclase and promotes proliferation of mouse primordial germ cells. Development 122 215–221.[Abstract]
Quinn P, Barros C & Whittingham DG 1982 Preservation of hamster oocytes to assay the fertilizing capacity of human spermatozoa. Journal of Reproduction and Fertility 66 161–168.[Abstract]
Redmer DA & Reynolds LP 1996 Angiogenesis in the ovary. Reviews of Reproduction 1 182–192.[Abstract]
Skinner MK 2005 Regulation of primordial follicle assembly and development. Human Reproduction Update 11 461–471.
Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH & Journot L 1993 Differential signal transduction by five splice variants of the PACAP receptor. Nature 365 170–175.[CrossRef][Medline]
Steiner AL, Parker CW & Kipnis DM 1972 Radioimmunoassays of cyclic nucleotides. Journal of Biological Chemistry 247 1106.
Van Blerkom J 2000 Intrafollicular influences on human oocyte developmental competence: perifollicular vascularity, oocyte metabolism and mitochondrial function. Human Reproduction 2 173–188.
Zhong Y & Kasson BG 1994 Pituitary adenylate cyclase-activating polypeptide stimulates steroidogenesis and adenosine 3', 5'-mono-phosphate accumulation in cultured rat granulosa cells. Endocrinology 135 207–213.[Abstract]
Received in final form 14 June 2006
Accepted 17 July 2006
Made available online as an Accepted Preprint 11 August 2006
This article has been cited by other articles:
|
|
 |
|
  M. Barberi, B. Muciaccia, M. B. Morelli, M. Stefanini, S. Cecconi, and R. Canipari Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary Reproduction, August 1, 2007; 134(2): 281 - 292. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||
P < 0.001.