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Endocrinology, doi:10.1210/en.2003-1004
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Endocrinology Vol. 145, No. 4 2071-2079
Copyright © 2004 by The Endocrine Society

Effect of Pituitary Adenylate Cyclase-Activating Polypeptide and Vasoactive Intestinal Polypeptide on Mouse Preantral Follicle Development in Vitro

Sandra Cecconi, Gianna Rossi, Marzia Barberi, Lucia Scaldaferri and Rita Canipari

Department of Biomedical Sciences and Technologies (S.C., G.R.), Faculty of Medicine, University of L’Aquila, 67100 L’Aquila, Italy; Department of Histology and Medical Embryology (M.B., R.C.), Faculty of Medicine, University of Rome "La Sapienza," 00161 Rome, Italy; and Department of Public Health and Cell Biology (L.S.), Histology Section, University of Rome "Tor Vergata," 00173 Rome, Italy

Address all correspondence and requests for reprints to: Rita Canipari, Department of Histology and Medical Embryology, University of Rome "La Sapienza," Via A. Scarpa 14, 00161 Rome, Italy. E-mail: rita.canipari{at}uniroma1.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It is transiently expressed in preovulatory follicles and positively affects several parameters correlated with the ovulatory process. It has also been shown to be expressed in the interstitial tissue around primordial and preantral follicles. The aim of the present study was to investigate whether PACAP influences preantral follicle growth and differentiation. Mouse preantral follicles were cultured for 5 d in the presence of FSH and increasing concentrations of PACAP or vasoactive intestinal polypeptide (VIP) (10-12 to 10-7 M). In the presence of FSH, follicles increased in diameter and formed an antrum. At the concentrations tested, neither PACAP alone nor VIP alone had any effect on follicle development, but the addition of either peptide to FSH-stimulated follicles caused a dose-dependent inhibition of follicle growth, antrum formation, granulosa cell proliferation, and estradiol production. The effect of PACAP on follicle growth and antrum formation was directly correlated with the length of stimulation and was reversible. Although exposure of follicles to 10-7 M PACAP and VIP did not affect oocyte growth, it severely impaired completion of meiotic maturation in oocytes isolated from the follicles and cultured for 17 h in medium alone. The cyclic production of PACAP by preovulatory follicles during the estrous cycle in adult rats and its induction by LH in the rat and mouse ovary suggest that this peptide may play a role in the local regulation of preantral follicle growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PITUITARY ADENYLATE CYCLASE-ACTIVATING polypeptide (PACAP), originally isolated from ovine hypothalamus, exists in two forms: PACAP-38 and PACAP-27, which share the same N-terminal 27 peptides, derived from tissue-specific proteolytic processing of the 176-amino acid precursor protein (1). The name of these two peptides reflects their potent action in stimulating cAMP production in anterior pituitary cells (2). On the basis of sequence similarity, PACAPs were included in the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family of peptides. They in fact share a 68% homology with VIP and 41% with GH-releasing factor, two other members of the same family (3). PACAP and VIP act on target cells through three different types of receptors: PACAP type I receptor (PAC1-R), which specifically binds to both PACAPs and VIP although with lower affinity, and to two others called VPAC1-R (4) and VPAC2-R (5), which bind to PACAPs and VIP with equally high affinity. It has been demonstrated that all three receptors are coupled to the adenylate cyclase pathway, but only PAC1-R can also be coupled to phospholipase C pathway (6). In addition to the central nervous system, PACAPs, VIP, and their receptors have been found in various organs and peripheral tissues such as lung, testis, adrenal, and ovary (7, 8, 9, 10), which suggests that such peptides may not play an exclusively neuroendocrine role. Moreover, PACAP (11) and PAC1-R (12, 13) are transiently produced by gonadotropin-stimulated preovulatory follicles.

Successful oocyte maturation, fertilization, and embryonic development depends on a correct, coordinated growth and development of the ovarian follicle. Follicular growth and differentiation is a process characterized by morphological and functional changes regulated primarily by FSH and LH. Besides being influenced by gonadotropins, follicles are also influenced by intra and extraovarian factors. Several studies have demonstrated that peptides such as PACAP and VIP influence important follicular functions. In fact, PACAP and VIP are involved in the regulation of steroidogenesis (14, 15), cAMP accumulation (16, 17), plasminogen activator production (18, 19), and oocyte maturation (17, 20). Moreover, PACAP and VIP contribute to the survival of granulosa cells by inhibiting apoptosis in preovulatory follicles (21, 22). Besides its location and effects in the preovulatory follicle, PACAP has also been found in theca/interstitial cells of follicles regardless of the stage of their development (21), whereas immunoreactive VIP has been identified in nerve fibers in developing follicles of rodent (15), bovine (23), and avian (24) ovary. On the basis of these observations, in the present study, we used an in vitro preantral follicle culture system to investigate the possible role of PACAP and VIP in the development of preantral follicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Swiss CD1 mouse families (Charles River, Calco, Italy) were housed in a temperature- (22–23 C) and light-controlled room (12 h lighting schedule, from 0600 to 1800 h). The animals were kept in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory animals. The experimental protocols were approved by the University Committee on Animal Care and Use.

Reagents
PACAP-38 and VIP were purchased from Calbiochem (San Diego, CA). Equine CG and hCG were purchased from Intervet (Livorno, Italy). All other reagents were obtained from Sigma Chemical (St. Louis, MO). Highly purified ovine FSH (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-o-FSH-19-SIAFP,BIO) was kindly provided by Dr. A. F. Parlow (National Hormone and Pituitary Program, NIH).

Preantral follicle isolation and culture
Individual preantral follicles were mechanically dissected, together with a small clump of thecal stromal tissue attached, from the ovaries of 24- to 26-d-old mice. Follicles were measured with a precalibrated ocular micrometer at magnification x40; those measuring 160 ± 10 µm in diameter (excluding thecal tissue) with a centrally placed spherical oocyte (mean diameter: 65 ± 1 µm) and no signs of somatic cell degeneration were chosen for further culture. Follicles were individually placed in 96-U-well microtitre-plates in 25 µl {alpha}MEM supplemented with 1% ITS (insulin 5 µg/ml; transferrin 5 µg/ml; sodium selenite 5 ng/ml), antibiotics (penicillin, 100 U/ml; streptomycin, 100 mg/ml), and 5% fetal calf serum (FCS). The medium was overlaid with 70 µl sterile mineral oil (embryo tested, d = 0.84 g/ml). Cultures were carried out for 5 d at 37 C in 5% CO2 atmosphere.

In another set of experiments, the follicles, cultured in the presence of FSH plus 10-7 M PACAP, were transferred into 25-µl drops of fresh culture medium supplemented with FSH alone at specific times from the beginning of culture (i.e. 48, 72, and 96 h); the follicles were all maintained in culture for a total of 6 d. Yet another series of experiments was conducted in which follicles were cultured in the presence of FSH and then transferred into 25-µl drops of FSH plus 10-7 M PACAP every 24 h starting from d 1. In a final series of experiments, follicles were cultured in the presence of 0.5 mM (Bu2)cAMP, a cAMP analog, with or without 10-7 M PACAP or VIP.

Culture medium was changed every other day, and spent medium was collected and stored at -80 C until assayed for 17ß-estradiol (E2).

Evaluation of follicle and oocyte growth and development
Follicle diameter was monitored daily using a calibrated micrometer. At the end of the culture period, the formation of an antral cavity was determined by the presence of a visible translucent area inside the follicle. All cultured follicles were carefully opened; the oocytes that were released, after being mechanically isolated from the surrounding cumulus cells, were measured and their meiotic stage was evaluated. Oocytes were either fixed immediately in 3% paraformaldeyhde/PBS or allowed to mature for 16–18 h in DMEM supplemented with 0.23 mM sodium pyruvate and 5% FCS, and then fixed.

Analysis of meiotic maturation
The stage of oocyte meiotic maturation was determined in germinal vesicle (GV)-arrested and in vitro-matured oocytes by Hoechst 33358 staining (5 µg/ml). Meiotic arrest was indicated by the presence of the GV and nucleolus. The analysis of the GV stages was performed by identifying GVs with an unrimmed nucleolus or either a partially or completely rimmed nucleolus, as previously described (25). Germinal vesicle breakdown (GVBD) and the appearance of the first polar body (PB) served, respectively, as markers for resumption of meiosis and complete oocyte nuclear maturation.

Morphological analysis of granulosa cell apoptosis
Granulosa cell apoptosis was evaluated as previously described (26). Briefly, on the fifth day of culture, follicles were mechanically dissected and the granulosa cells were released in the medium. Cells from single follicles were fixed for 15 min in 3% paraformaldeyhde/PBS and cytocentrifuged onto a glass slide at 200 x g for 10 min. The samples were washed three times with PBS and the chromatin was stained using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method according to the manufacturer’s instructions (Apop Tag, S7100-Kit, Oncor, Gaithersburg, MD). Apoptotic cells were identified and counted in three or more randomly selected fields with at least 100 cells each.

Granulosa cell proliferation
DNA content was determined as previously reported (26). Briefly, DNA extracted from pools of six follicles per assay on day 0 and 5 of culture was measured by fluorometric assay using Hoechst 33258 (0.1 mg/ml) as a fluorescent dye. Aliquots of samples were added to 2 ml of dye solution and immediately measured by fluorometer (Perkin-Elmer, Milan, Italy) at 365/460 nm (excitation/emission) wavelengths. DNA was expressed as nanograms per follicle, and dilutions of sonicated salmon sperm DNA were used as a standard.

E2 determination
The concentration of E2 in the medium was determined by means of a RIA kit, according to the procedure described by the manufacturer (Radim, Pomezia, Italy), as previously reported (27). The intraassay and the interassay coefficients of variation were 4 and 5%, respectively. The RIA results were analyzed with a program that uses a four-parameter logistic function, and unknowns were interpolated from the resultant curve.

Assay of cAMP
Individual preantral follicles were cultured for 3 h in {alpha}MEM with 5% FCS in the presence of the various hormones. At the end of incubation, follicles and media were collected, homogenized in 10% trichloroacetic acid, and centrifuged. The supernatant was extracted three times with two volumes of water-saturated diethyl ether.

The amount of cAMP present was measured by RIA (28). Samples were acetylated before the assay, according to the procedure of Harper and Brooker (29). 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%. Standard curve was calculated using a log-linear curve fit with %B/Bo (y-axis) against cAMP concentrations (x-axis). Values were normalized to milligrams of proteins present in the sample and were expressed as fold induction of the control arbitrarily set as 1. Protein content was measured according to the method of Lowry et al. (30) using BSA as the standard.

RNA isolation and analysis by means of RT PCR
Total cellular RNA was extracted from preantral follicles cultured for 5 d under various conditions using the acid guanidinium thiocyanate-phenol-chloroform method (31). The purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before the analytical procedures were carried out.

Levels of aromatase mRNA were determined by means of the RT-PCR method. Total RNA (0.5 µg) were reverse transcribed, in a final volume of 20 µl, using 200 U cloned M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) in the presence of 2.5 µM random hexamers and 1 mM deoxynucleotide triphosphate for 1 h at 37 C and then heat denatured for 5 min at 95 C; for each sample, half of the resulting cDNA was subjected to PCR using primers designed for the amplification of aromatase (upstream, 5'-GCACGAGAATGGCATCAT-3'; downstream, 5'-GTTAGAAGTGTCCAGCATG-3'; amplified product, 200 bp) (32) and the ß actin housekeeping gene (upstream, 5'-TGTGCTGTCCCTGTATGCC-3'; downstream, 5'-TCGTTGCCAATAGTGATGAC-3'; amplified product 344 bp) (33). cDNAs were coamplified using a DNA thermal cycler (PerkinElmer/Cetus) and the Taq DNA polymerase (2 U/tube) with 15 pmol of both upstream and downstream primers and 2.2 mM magnesium chloride in a final volume of 50 µl that was overlaid with 25 µl mineral oil. Thirty-five cycles (60 sec at 94 C, 60 sec at 60 C, and 60 sec at 72 C with a 10-min final extension) were applied for the amplification of aromatase cDNA. Primers for the housekeeping gene were added to the PCR tubes during the denaturation step in the 10th cycle. In these conditions the plateau for the housekeeping amplification was not reached. For each sample, 25 µl PCR amplification product were analyzed on 2% agarose gel and stained with ethidium bromide. Standard DNA molecular weight (Ladder VI, Roche Molecular Biochemicals, Indianapolis, IN) was run to provide the appropriate size marker. Amplified products were quantitated by means of computer analyses, and aromatase mRNA levels were normalized against actin.

For the analysis of PACAP/VIP receptor mRNAs, total RNA was extracted from 14-d-old animal whole ovaries as described above. Mouse brain was used as a positive control tissue. Reverse transcription was performed on 1 µg total RNA using 200 U M-MLV reverse transcriptase (Invitrogen) in the presence of 500 µg/ml oligo dT primers and 0.5 mM deoxynucleotide triphosphate, for 1 h at 37 C and then heat denatured for 15 min at 70 C; aliquots of the resulting cDNA were subjected to PCR using primers designed for amplification of PAC1-R (upstream, 5'-CAA GAA GGA GGA GCA AGC CAT GTG C-3'; downstream, 5'-CAT CGA AGT AAT GGG GAA GGA GGG-3'; amplified product, 317 bp), VPAC1-R (upstream, 5'-TGA GCC TGT TCA GGA AGC TGC ACT-3'; downstream, 5'-CTC GAA TAT GGG CTG CTA TCA TTC TT-3'; amplified product, 526 bp) (34), VPAC2-R (upstream, 5'-TCT GCC TCT TCA GGA AGC TGC ACT-3'; downstream, 5'-ATT CCG GGA CAT GGA CCA GCT GT-3'; amplified product, 797 bp) and S16 ribosomal RNA (upstream, 5'-TCC AAG GGT CCG CTG CAG TC-3'; downstream, 5'-CGT TCA CCT TGA TGA GCC CAT T-3'; amplified product, 139 bp). cDNAs were amplified using a DNA thermal cycler (Eppendorf, Hamburg, Germany) and 2 U/tube of Taq DNA Polymerase (Roche Molecular Biochemicals) with 1 pmol of both upstream and downstream specific primers and 1.25 mM magnesium chloride in a 25 µl reaction volume. PCR was performed for 45 amplification cycles for PAC1-R, VPAC1-R, and VPAC2-R, and for 20 cycles for S16 under the following conditions: 30 sec at 94 C, 30 sec at 60 C, and 40 sec at 72 C with a 4-min final extension. For each sample, 10 µl PCR amplification product were analyzed on 2% agarose gel stained with ethidium bromide and quantitated by means of computer analyses. mRNAs for the three receptors were normalized against S16.

Controls for DNA contamination or PCR carry-over were performed without the M-MLV reverse transcriptase or the RNA during reverse transcription.

Statistical analysis
Statistical analyses were performed by ANOVA followed by the Tukey-Kramer test for comparison of multiple groups. Values with P < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PACAP and VIP on in vitro follicle growth and development
The effects exerted on preantral follicle development by PACAP and VIP were evaluated during a 5-d culture period. To this end, follicles were incubated in medium alone (C), 10-7 M PACAP 38, 10-7 M VIP, or 100 ng/ml FSH either with or without PACAP or VIP. As already shown (26), the addition of FSH resulted in follicle growth from the preantral (160 ± 10 µm) to preovulatory stages (about 400 µm). By contrast, no significant diameter increase was observed in follicles cultured in medium without gonadotropin (Fig. 1Go). Although neither PACAP nor VIP alone promoted follicle growth, they did severely affect growth of preantral follicles cultured in the presence of FSH. In fact, by the end of culture, the mean diameter of follicles cultured in the presence of FSH was approximately 400 µm, whereas in the presence of FSH plus PACAP or VIP it was approximately 290 and 295 µm, respectively (i.e. about 75% of that of follicles stimulated by FSH alone) (Fig. 1Go).



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  		FIG. 1. Changes in the diameter of preantral follicles from immature mice. Preantral follicles were cultured for 5 d in medium containing 2% FCS (C), or supplemented with FSH (100 ng/ml), PACAP (10-7 M), VIP (10-7 M), FSH+PACAP, or VIP. Follicular growth was checked daily. Values represent the mean ± SEM of three to five independent experiments. The total number of follicles examined was between 200 and 400 for each point. *, P < 0.01 PACAP vs. FSH+PACAP; **, P < 0.001 FSH vs. FSH+PACAP.

 
On d 5 of culture, no antrum formation was observed in the control or PACAP- or VIP-treated follicles, whereas approximately 80% of the FSH-treated follicles developed large antral cavities. The addition of either peptide to FSH-stimulated follicles caused a dose-dependent inhibition in the percentage of antrum formation (Fig. 2Go). In view of these results, before any further evaluation was conducted, follicles were divided into two groups on the basis of their capacity to develop antrum.



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  		FIG. 2. Dose-dependent effect of PACAP and VIP on FSH-stimulated antrum formation. Preantral follicles were cultured for 5 days in medium containing 2% FCS (C) or supplemented with FSH (100 ng/ml), FSH + PACAP, or VIP at different doses (10-13 to 10-7 M). Values represent the mean ± SEM of three to five independent experiments. *, P < 0.01, **, P < 0.001 vs. FSH.

 
The pattern of E2 release was closely related to antrum development, with FSH stimulation determining antrum formation in the majority of follicles and causing a significant increase in steroid production. Significantly lower levels of estradiol per follicle were observed in conditions that did not support antrum formation, i.e. control follicles (C) and the majority of follicles cultured with FSH together with either PACAP or VIP (Fig. 3Go, upper panel). It is noteworthy that the few follicles that did develop an antral cavity in response to FSH, despite the presence of the peptides, displayed estradiol levels similar to those observed in the FSH-only-treated follicles (data not shown).



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  		FIG. 3. Changes in E2 (upper panel) and DNA content (lower panel) in follicles cultured for 5 d with medium containing 2% FCS (C), FSH (100 ng/ml), FSH + PACAP (F+P), or VIP (F+V) (10-7 M). Values represent the mean ± SEM of three independent experiments. *, P < 0.05, **, P < 0.001 vs. C.

 
Similar results were obtained when DNA content per follicle was measured. The decline in follicle growth and estradiol production observed in follicles cultured in the presence of FSH and PACAP or VIP was accompanied by a decrease in granulosa cell proliferation evaluated on the basis of the DNA content (Fig. 3Go, lower panel). The few follicles that did form an antrum in response to FSH, despite the presence of the two peptides, displayed higher levels of DNA content also in this case (data not shown).

The inhibition of follicle growth was not dependent on increased apoptosis. In fact, although PACAP and VIP did not reduce the rate of apoptosis in granulosa cells if compared with control follicles, FSH did so significantly, regardless of the addition of the peptides (Fig. 4Go).



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  		FIG. 4. Percentage of apoptosis in granulosa cells obtained from preantral follicles cultured for 5 d with medium containing 2% FCS (C) or supplemented with FSH (100 ng/ml), PACAP (10-7 M), VIP (10-7 M), FSH + PACAP (F+P), or VIP (F+V). Values represent the mean ± SEM of four independent experiments. *, P < 0.05 vs. C.

 
Effect of FSH and PACAP on aromatase activity
The effect of PACAP (10-7 M) on FSH (100 ng/ml) modulation of aromatase mRNA levels in follicles cultured for 5 d was assessed by means of semiquantitative RT-PCR. Densitometric analysis of the amplified product showed that FSH increased the level of aromatase mRNA (Fig. 5Go); this increase was, however, associated with the presence of antrum formation as previously observed for E2 production. In fact, the small proportion of follicles that did not respond properly to FSH and failed to form an antrum displayed significantly lower levels of aromatase-mRNA (data not shown). PACAP alone did not have any effect on the levels of aromatase-mRNA, whereas it significantly inhibited the FSH-stimulated mRNA in the majority of treated follicles (Fig. 5Go). In accordance with the data obtained for estradiol production, the few follicles that did develop antra in response to FSH, despite the presence of PACAP, displayed estradiol levels similar to those observed in the FSH-only-treated follicles (data not shown).



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  		FIG. 5. Effect of PACAP on FSH-induced aromatase mRNA level in preantral follicles cultured for 5 d either with or without FSH in the absence or presence of PACAP(10-7 M). At the end of the culture period, total RNA was extracted, and aromatase and actin mRNAs levels were assessed by RT-PCR. Amplification products were separated on 2% agarose gel and stained with ethidium bromide. The bands were analyzed with a chemiluminescence detection system (Raytest, Straubenhart, Germany). Aromatase values are normalized by their respective actin values and represent the mean ± SEM of two independent experiments. **, P < 0.01, ***, P < 0.001 vs. C.

 
Effect of PACAP on cAMP levels
To evaluate whether PACAP stimulates cAMP accumulation, preantral follicles with a diameter of 160 µm were cultured for 3 h in medium alone (basal), FSH (100 ng/ml), or PACAP (10-7 M) in the presence or absence of FSH. Stimulation with FSH and PACAP alone significantly increased the levels of total cAMP. An additive effect on cAMP production was observed when PACAP was used in combination with FSH (Table 1Go).


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  		TABLE 1. Effect of FSH, PACAP, and PACAP plus FSH on cAMP accumulation by follicles cultured for 3 h

 
Effect of PACAP and VIP on cAMP-induced antrum formation
To evaluate whether the negative effect exerted by PACAP and VIP on antrum development occurred downstream of cAMP production, preantral follicles were incubated in the presence of (Bu2)cAMP (0.5 mM), with or without 10-7 M PACAP or VIP. The results (Table 2Go) show that antrum formation was stimulated by (Bu2)cAMP and that the presence of 10-7 M PACAP significantly impaired (Bu2)cAMP-stimulated antra formation, whereas that of VIP was ineffective.


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  		TABLE 2. Effect of PACAP on the percentage of cAMP-induced antrum formation on d 5 of culture

 
Morphological evaluation and meiotic competence of in vitro-grown oocytes
To evaluate whether PACAP and VIP affect oocyte growth and/or competence to mature up to MII, oocytes from in vitro-grown follicles were collected on d 5 of culture, their final diameter was measured; half were fixed for GV configuration analysis and half allowed to mature in vitro for 17 h. Oocyte diameters significantly increased from 62 ± 1 to 74 ± 1 µm (P < 0.001) in all the culture conditions tested. The proportion of oocytes that underwent maturation was not correlated with antrum formation. In fact, although follicles failed to form antra in the absence of FSH, the percentage of GVBD and Met II oocytes obtained from those follicles was comparable with that obtained from FSH-treated follicles. The ability to resume meiosis was low both in follicles treated with PACAP alone and in those treated with PACAP and FSH that did not form antra (Fig. 6Go, upper panel). As already stated for the other parameters considered in this study, the proportion of oocytes obtained from the few follicles that escaped PACAP inhibition and resumed meiosis was similar to that of FSH-treated follicles. However, the ability to complete meiosis up to MII was severely affected by PACAP regardless of antrum development (Fig. 6Go, lower panel). In fact, less than 50% of oocytes obtained from the few follicles that did form antrum in the presence of PACAP extruded the first PB, and this percentage was even lower (25%) in those from follicles without an antrum. VIP was significantly less effective and its inhibitory effect was observed only in those follicles that did not form antra.



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  		FIG. 6. Maturation competence of oocytes obtained from follicles cultured for 5 d with medium alone (C), FSH (100 ng/ml), PACAP (10-7 M), VIP (10-7 M), FSH+PACAP, or VIP. At the end of the culture period, oocytes obtained from follicles with or without antra were separated and subjected to in vitro maturation for 17 h. Values represent the mean ± SEM of four independent experiments (150–200 oocytes/group). Upper panel, Percentage of oocytes that underwent GVBD. Lower panel, Percentage of oocytes that underwent GVBD and exhibited a PB (MII). There are no significant differences between groups with the same superscript, whereas groups with different superscriptsare significantly different.

 
This impaired ability to complete meiotic maturation was accompanied by a more immature stage of GV maturation, as is demonstrated by the fact that we found a slightly higher percentage of GVs with an unrimmed nucleolus in follicles cultured in conditions that did not support antrum formation (FCS, PACAP, and VIP alone, FSH without antrum) (data not shown).

Modulation of PACAP responsiveness
To evaluate whether PACAP inhibition could be reversed by peptide withdrawal from the culture medium, follicles were cultured in the presence of FSH plus 10-7 M PACAP and, at specific times from the beginning of culture (i.e. 48, 72, and 96 h), were transferred into 25-µl drops of fresh culture medium supplemented with FSH alone; the follicles were all maintained in culture for a total of 6 d. The results (Fig. 7Go, upper panel) show a increase in follicle growth that inversely correlates with the length of PACAP stimulation. A significant decrease in the inhibition of antrum formation was evident only when PACAP was removed within the first 2 d of culture.



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  		FIG. 7. Reversibility of PACAP inhibitory effect on antrum formation (upper panel). Follicles were treated for 6 d with 100 ng/ml FSH alone (FSH) or with FSH and 10-7 M PACAP for 2 (0–2), 4 (0–4), and 6 (0–6) d and then washed and further cultured in medium with FSH alone. Follicles were maintained in culture for a total of 6 d. The effect of PACAP added at different culture times on antrum formation (lower panel). Follicles were cultured with 100 ng/ml FSH before 10-7 M PACAP was added on the first, second, and fourth days of culture. Follicles were cultured for a total of 6 d. Values represent the mean ± SEM of three independent experiments. **, P < 0.01, ***, P < 0.001 vs. respective FSH.

 
To evaluate a temporal window within which PACAP could affect follicle growth and antrum formation, follicles were maintained from the beginning of culture in the presence of FSH and then transferred into 25-µl drops of FSH plus 10-7 M PACAP at 48-h intervals. As shown in Fig. 7Go (lower panel), the inhibitory effect of PACAP on follicle growth was directly correlated with the length of stimulation, with antrum formation being affected only when added at the beginning of culture.

PACAP and VIP receptors
To characterize the type of PACAP receptor present in juvenile mouse ovary, total RNA was extracted from whole 14-d-old mice ovaries and analyzed by RT-PCR. This age was chosen to obtain ovaries with only primordial, primary and preantral follicles. A positive signal for VPAC1-R and VPAC2-R appeared after 35 cycles of amplification, whereas that for PAC1-R was very weak (Fig. 8Go).



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  		FIG. 8. Expression of different molecular forms of PACAP/VIP receptor subtypes as detected by RT-PCR in 14-d-old mouse ovary. Total RNA was subjected to RT-PCR using primers specific for PAC1-R, VPAC1-R, and VPAC2-R receptors, as reported in Materials and Methods. An aliquot of each PCR product and DNA molecular weight markers (100-bp DNA ladder, Promega) were electrophoresed on 1.5% agarose gel and stained with ethidium bromide. No signal was detected in the negative control for either set of primers. The predicted fragment lengths for the three different receptor mRNAs are 317 bp for PAC1-R (lane 1), 526 bp for VPAC1-R (lane 2), and 797 bp for VPAC2-R (lane 3). Lane 4 represents the standard markers. The figure is representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The contemporary production of PACAP (21) and its receptor (13) in response to gonadotropins in rat preovulatory follicles, together with the positive effects exerted by PACAP on parameters linked to ovulation, provide strong evidence that PACAP may play a role in the complex physiology of the follicle nearing ovulation. A similar role has been hypothesized for VIP because VIP has been shown to stimulate ovulation in perfused rat ovaries (35). However, in the rat, PACAP and VIP immunoreactivity has also been observed around primordial and small preantral follicles (11) as well as in the theca layers of developing follicles (15), which thus points to an additional role of these two peptides in the regulation of early folliculogenesis.

Although no data are available on the production of PACAP and VIP in juvenile mouse ovary, our data show the presence of the PACAP and VIP receptors. The main receptor forms in juvenile mouse ovaries are VPAC1-R and VPAC2-R, whereas the presence of PAC1-R is negligible. However, we have preliminary results showing an increase of PAC1-R expression in mouse ovaries that correlates with age increase. Moreover, stimulation of PACAP mRNA has been shown to occur in whole mouse ovaries after the LH surge (36). These findings therefore, strongly suggest that PACAP and VIP also play a role in mouse ovary.

In the present study, we demonstrate that PACAP and VIP inhibit several follicular parameters correlated with normal follicle development, such as growth, antrum formation, and estradiol production. However, the observation that a small proportion of cultured follicles does not behave like rest of the follicles (i.e. they do not form antra in response to FSH or do form antra in response to FSH, despite the presence of PACAP or VIP) suggests that the population of retrieved preantral follicles is heterogeneous and responds in different ways to the same stimulus.

The increase in follicle size depends on a balance between an increase in the number of granulosa cells (37) and the levels of apoptosis (38). In our culture conditions, we observed a diminished rate of granulosa cell proliferation not associated with increased apoptosis. In fact, apoptotic frequency was less than 10% even in the absence of FSH, and the addition of PACAP and VIP did not decrease cell survival. Besides a diminished rate of cell proliferation, we also observed, in those follicles that did not form antra, a decreased aromatase activity, which, combined with the former, led to a decreased E2 production per follicle. Although granulosa cell proliferation and differentiation during the development of preovulatory follicles in vivo is modulated by FSH and estradiol (39, 40, 41), it has been shown that the absence of estradiol in follicles cultured in vitro in the presence of an aromatase inhibitor does not affect either follicle or oocyte growth (42). Therefore, the decreased production of estradiol per follicle observed in our conditions might be a sign of the state of the follicle rather than the cause of follicle growth arrest.

A direct effect of PACAP on granulosa cell proliferation may be hypothesized because PACAP has been shown to modulate cell proliferation in different systems. PACAP not only stimulates primordial germ cell proliferation (43) but also inhibits cell proliferation in the development of mouse neural tube, in which it is modulated by the presence of fibroblast growth factors and other growth factors (44, 45). Moreover, PACAP and VIP both inhibit cell proliferation in a murine cell line of neuroblastoma (46). However, the mechanism underlying the inhibitory effect of PACAP is not yet fully understood. In many systems the action of PACAP is mediated by an increase in cAMP production, and our data indeed show that PACAP alone stimulates cAMP production and amplifies FSH stimulation of cAMP. However, correct gene expression in granulosa cells is dependent not only on an increase in the level of cAMP but also on its intensity and duration. It has been shown that FSH and low levels of cAMP promote granulosa cell proliferation and that the LH surge and high levels of cAMP instead induce the exit from the cell cycle (40). Moreover, perturbing cAMP signals by altering its intensity and duration can interfere with the differentiative program of granulosa cells (36) as well as in a prostate cancer cell line (LNCaP) (47).

However, the fact that PACAP can also inhibit (Bu2)cAMP-stimulated antrum formation suggests that the inhibitory action of this peptide occurs downstream of cAMP production.

In addition to the stimulation of cAMP, an alternative hypothesis is that PACAP, through the interaction with its receptor PAC1, may activate phospholipase C and inositol 1,4,5-triphosphate signaling. This hypothesis is supported by the fact that VIP, which does not bind PAC1-R and cannot consequently activate phosphoinositide turnover, does not inhibit cAMP-induced antrum formation.

Although oocyte growth rate was not affected by the different follicle treatments, our results demonstrate that fewer oocytes reached the Met II stage when follicles were cultured in the presence of PACAP. This negative effect does not appear to be a direct effect of the peptide on the oocyte itself because PACAP accelerates oocyte maturation both in rat (19) and mouse oocyte-cumulus cell complexes (our unpublished results). This impairment may therefore be ascribable to an effect on the whole follicle. One possible explanation is that PACAP promotes an inappropriate differentiation of granulosa cells as previously described for FSH and insulin (48). Indeed, PACAP has been shown to play a role in periovulatory progesterone production, which suggests its involvement in luteinization of rat granulosa cells (14). We can therefore hypothesize that PACAP influences the rapid switch from the highly proliferative stage to the nonproliferative, terminally differentiated phase of luteal cells. Whereas this effect is positive in the case of preovulatory follicles, it may be deleterious in the case of preantral follicles and consequently be deleterious to the development of oocytes in those follicles.

Because we were not able to demonstrate PACAP production in juvenile mouse ovaries in this paper, we cannot conclude that PACAP influences the first wave of growing follicles. However, whereas we are fully aware of the fact that follicles obtained from immature or mature cycling mice might respond differently to the same stimulus and that this work was performed using follicles obtained from immature mice, and not cycling mice, it is noteworthy that our follicles respond to FSH similarly to those obtained from cycling animals (49). We may, consequently, hypothesize that in the adult cycling mouse, in which PACAP is transiently expressed in the morning of estrus by preovulatory follicles (11), PACAP inhibits FSH-induced growth of small preantral follicles. This hypothesis, moreover, is in accordance with the fact that the development rate of follicles in adult cycling rodents is slower than that of the first wave of follicles in juvenile animals (50).

In conclusion, our results suggest that PACAP and VIP are deeply involved in the ovarian physiology and that they can, at least in the adult ovary after the first LH surge, modulate FSH-dependent growth of developing follicles while acting synergistically with LH on the differentiation of preovulatory follicles.


   Acknowledgments
 
We thank Mr. S. Greci for his excellent technical assistance, Dr. A. F. Parlow and the National Hormone and Pituitary Distribution Program (NIDDK) for providing the ovine FSH and LH, and Mr. Lewis Baker for reviewing the English in the manuscript.


   Footnotes
 
This work was supported by grants from the Ministero per l’Università e la Ricerca Scientifica (60% and co-fin 2000–2001 to R.C.; 60% to S.C.).

Abbreviations: E2, 17ß-Estradiol; FCS, fetal calf serum; GV, germinal vesicle; GVBD, germinal vesicle breakdown; PACAP, pituitary adenylate cyclase-activating polypeptide; PB, polar body; VIP, vasoactive intestinal polypeptide.

Received August 5, 2003.

Accepted for publication December 24, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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