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Regular Article |
a Department of Clinical Biochemistry, University of Copenhagen, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark
ABSTRACT
Pituitary adenylate cyclase-activating peptide (PACAP) is transiently expressed in ovarian granulosa/lutein cells from eCG/hCG-treated rats, and in vitro immunoneutralization of endogenously released PACAP inhibits acute progesterone secretion and subsequent luteinization in such cells. This suggests that PACAP mediates locally some of the effects of the LH surge, but the putative PACAP receptor(s) involved in such an auto or paracrine activity is presently unknown. Reverse-transcription polymerase chain reaction with specific primers to the three cloned PACAP-binding receptors called PAC1, VPAC1, and VPAC2 demonstrated both PAC1 and VPAC2 mRNA in extracts from preovulatory follicular cells. Radioligand-binding assays revealed the presence of high-affinity binding sites with characteristics of these two receptors on the intact cells, and autoradiography demonstrated that the binding was restricted to a minor proportion of the follicular cells as well as the oocytes. Pituitary adenylate cyclase-activating peptide and vasoactive intestinal peptide (VIP) dose-dependently stimulated cAMP accumulation and acute progesterone accumulation. Forskolin and db-cAMP also stimulated acute progesterone accumulation, and the protein kinase A inhibitor H89 dose-dependently inhibited peptide induced acute progesterone accumulation, suggesting involvement of cAMP and the protein kinase A pathway in the process. In conclusion, two of the three PACAP binding receptors are present on preovulatory follicular cells and are involved in the effects of PACAP on acute progesterone production. The data provide further evidence to establish PACAP as an auto- or paracrine regulator of LH-induced acute progesterone production in rat preovulatory follicles.
cAMP, corpus luteum, corpus luteum function, neuropeptides/neurotransmitters, ovum, signal transduction
INTRODUCTION
The midcyclic gonadotropin surge is the obligatory signal for the biochemical cascade that ultimately leads to ovulation and subsequent luteinization of the ruptured follicles in the ovary. Multiple ovarian genes are expressed and participate in the local regulation of periovulatory events, but the complex interactions between locally produced substances and the gonadotropins are still only partially understood [1, 2].
Pituitary adenylate cyclase-activating peptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family [3, 4], has recently emerged as a potential local regulator of various aspects of ovarian physiology [5–12] and of preovulatory events in particular [13–15]. A few hours following the gonadotropin surge, PACAP is transiently expressed in theca cells and granulosa/lutein cells of large preovulatory follicles from both adult cyclic [13] and eCG/hCG-stimulated immature ovaries [13, 15]. Exogenous PACAP stimulates acute progesterone production in granulosa/lutein cells stimulated in vivo with eCG and hCG and immunoneutralization of endogenously released PACAP inhibits acute progesterone production and subsequent luteinization in such cells [14]. In preovulatory follicles stimulated in vivo with eCG, PACAP stimulates progesterone production and suppresses apoptosis, and furthermore, a PACAP antagonist inhibits LH-induced suppression of apoptosis [15]. These findings suggest that PACAP is an autocrine or paracrine regulator in the preovulatory ovary, mediating some of the effects of the LH surge.
The putative PACAP receptor(s) involved in these activities is presently unknown. Three different PACAP receptors belonging to the seven-transmembrane-spanning, G-protein-coupled family of receptors have been cloned in the rat [4, 16]. One called PAC1 (also known as PVR1 or PACAP type 1 receptor) exists in a basic form and in at least five different splice variants that either contain or lack each of two alternative exons named hip and hop [17]. The two others are called VPAC1 (also known as VIP1, PVR2, or PACAP type 2 receptor) [18] and VPAC2 (also known as VIP2, PVR3, or PACAP type 3 receptor) [19]. The mRNA from PAC1 and VPAC2 has been demonstrated in ovarian tissue by Northern blot, reverse-transcription polymerase chain reaction (RT-PCR), or in situ hybridization [6, 11, 12, 20]. Pituitary adenylate cyclase-activating peptide binding to ovarian tissue membranes has also been reported [21]. At present, however, the characteristics, the cellular localization, and possible cycle-specific expression of PACAP receptors in the ovary have not been established
In this study, we extend our previous examination of isolated granulosa/lutein cells from preovulatory follicles by characterizing molecularly, pharmacologically, and functionally the PACAP receptors present on such cells and their involvement in the effects of PACAP on acute progesterone production.
MATERIALS AND METHODS
Animals
Immature female Wistar rats, 22–26 days old, were used. They were housed under standard laboratory conditions with free access to food and water and a 12L:12D cycle. They were injected s.c. with 15 IU eCG (Sigma, St. Louis, MO), followed 48 h later by intraperitoneal injection of 10 IU hCG (Profasi, Serono, Sweden). Animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. All protocols had the approval of the institutional committee on animal care and use at the University of Copenhagen.
Cells
Granulosa/lutein cells were harvested 4–6 h after the hCG injection. At this time, ovarian PACAP expression starts in vivo [14]. The animals were decapitated and the ovaries were removed aseptically, freed from adherent tissue, and transferred to a Petri dish with culture medium (McCoy 5A; Life Technologies, Paisley, Scotland, UK). Under a stereomicroscope, the 15–20 largest follicles from each ovary were punctured with a 27-gauge needle, and the ovaries were gently pressed to release the intrafollicular cells. The cells were centrifuged at 250 x g for 10 min, washed twice, and counted on a hemocytometer. Viability was always above 75%, as determined by the trypan blue exclusion method.
RNA Extraction and RT-PCR
Granulosa/lutein cells from 5 different animals were pooled. Total RNA, extracted by the guanidinum thiocyanate method [22], was dissolved in sterile water and quantified by optical density at 260 nm. A RT-PCR was performed with the Titan RT-PCR kit (Boehringer Mannheim, Mannheim, Germany) using 20 pmol of each specific primer and 0.2 µg total RNA according to the manufacturer's suggestions. 10 µl of the RT-PCR product was submitted to electrophoresis on an agarose gel (2%) and stained with ethidium bromide.
PAC1. Four primers (P1–P4) were used to amplify and identify the different splice variants of the PAC1 receptor mRNA, as described elsewhere [23]. These primers, which are based on the reported sequence of the receptor [17], are as follows: P1, 5'-CAT CCT TGT ACA GAA GCT GC-3' (sense primer located at position 984–1003, i.e., upstream of the insertion of the hip/hop cassettes); P2, 5'-CCT CAG ACC AGC ATT CAC C-3' (sense primer located at position 1100–1118, i.e., inside the hip cassette); P3, 5'-TCC ACC ATT ACT CTA CGG CT-3' (sense primer located at position 1198–1217, i.e., inside the hop cassette); and P4, 5'-GGT GCT TGA AGT CCA TAG TG-3' (antisense primer located at position 1437–1456, i.e., downstream from the insertion site of the hip/hop cassettes). The expected sizes of the RT- PCR products using primer pair Pl/P4 were as follows: 305 base pairs (bp) for the basic variant, 386 bp for the hop 2 variant, 389 bp for the hop 1 and the hip variants, and 473 bp for the hip-hop 1 variant. For P2/P4, these were 273 bp for the hip variant and 357 bp for the hip-hop 1 variant. For P3/P4, these were 256 bp for the hop 2 variant and 259 bp for the hop 1 and the hip-hop 1 variant.
VPAC1. The primers are based on the reported sequence of the VPAC1 receptor mRNA [18]. The sense primer is located at position 809–828, and its sequence is as follows: 5'-TCC GAG CGG AAG TAC TTC TG-3'. The antisense primer is located at 1148–1167, and its sequence is as follows: 5'-ACC TGG GCC TTG AAG TTG TC-3'. The expected size of the RT-PCR product is 359 bp.
VPAC2. The primers are based on the reported sequence of the VPAC2 receptor mRNA [19]. The sense primer is located at position 341–360, and its sequence is as follows: 5'-CAC TAG TGA TGG GTG GTC GG-3'. The antisense primer is located at position 704–723, and the sequence is as follows: 5'-GCC AGT AGA AGT TCG CCA TG-3'. The expected size of the RT-PCR product is 380 bp.
Receptor Ligand-Binding Assay and Autoradiography
Cells were diluted to 2.5 x 106 cells/ml in incubation buffer (Krebs/Ringer/Hepes with MgCl2 (1 M), CaCl2 (2 M), and Bacitracin (1 mg/ml). Aliquots of 100 µl were incubated in Eppendorf tubes for 1 h at room temperature. PACAP 27 and VIP (Peninsula, St. Helens, UK) were radiolabeled as described elsewhere [24, 25]. Twenty-five microliters of 125I-labeled PACAP 27 (2000 cpm/µl) or 125I-labeled VIP (2000 cpm/µl) were incubated with 25 µl incubation buffer as a control or with increasing concentrations of unlabeled PACAP 38 (Peninsula) or VIP diluted with incubation buffer. Incubations were terminated by the addition of 100 µl ice-cold incubation buffer followed by centrifugation at 2000 x g for 5 min at room temperature. After aspiration of the supernatants, the cell-bound tracer was measured in a gamma counter. Data are presented as specific 125I-labeled PACAP 27 or 125I-labeled VIP binding as a percentage of the control value, with nonspecific binding determined after addition of PACAP 38 (1 µM) or VIP (1 µM). Data are the mean ± SEM from five different experiments, each performed in duplicate.
Cells used for autoradiography were processed as described above. After the final centrifugation, the supernatants were aspirated, and the pellets were washed twice with ice-cold incubation buffer. The pellets were resuspended in 200 µl of Stefanini fixative (2% paraformaldehyde and 15% picric acid in 0.1 M phosphate buffer [pH 7.2]) and incubated for 24 h. The cell suspension was then smeared on a glass slide, dried at room temperature, dipped in Amersham LM-1 emulsion (Amersham Denmark, Copenhagen, Denmark), and exposed for 5 days before development. Finally, the specimens were stained with 1% eosin and mounted in Pertex. Various smearing methods were tested, but an uneven distribution of the cells and significant variations in the background-labeling activity were observed on individual glass slides in all experiments. This impedes an adequate and uniform definition of the specific labeling on the individual cells, and consequently, an exact quantification of the proportion of labeled cells could not be performed. At least six different experiments were performed, and more than 200 oocytes were examined.
cAMP Accumulation
Cells were diluted to 2.5 x 105 cells/ml in McCoy 5A medium. Aliquots of 200 µl were incubated in Eppendorf tubes for 30 min at room temperature with increasing concentrations of PACAP 38 or VIP diluted in the same culture medium (final concentrations, 10-10–10-6 M). Incubations were terminated and the total cAMP content was extracted by the addition of 200 µl ice-cold TCA (10%) and centrifugation at 2000 x g for 5 min. Three hundred milliliters of supernatant was neutralized with 100 µl Tris buffer (1 M) and was stored at -30°C until analysis in duplicate with a commercial RIA as specified by the producer (Amersham Denmark). Before analysis, samples were diluted 1:25 with assay buffer. Data are presented in picomoles per well and are the mean ± SEM from five different experiments, each performed in duplicate.
Progesterone Accumulation: Effects of PACAP 38 and VIP
Cells were diluted to 2.5 x 105 cells/ml in McCoy 5A culture medium supplemented with penicillin, streptomycin, and testosterone (10-7 M; Sigma; final ethanol concentration, 0.01% v/v). Aliquots of 200 µl (5 x 104 cells per well) were placed in 96-multiwell dishes (Nunc, Copenhagen, Denmark). A dose-response study was performed with increasing concentrations of PACAP 38 and VIP (final concentrations, 10-10–10-6 M) diluted in culture medium. The cells were incubated for 2 h at 37°C in 5% CO2 and 95% atmospheric air, and incubations were terminated by aspiration of the cell suspension, followed by centrifugation at 2000 x g for 5 min at room temperature. The supernatants were stored at -20°C and later analyzed for progesterone with a commercial RIA kit (Orion Diagnostica, Espoo, Finland). Data (in nanomoles per liter) are presented as the mean ± SEM from six different experiments, each performed in duplicate.
Progesterone Accumulation: Influence of cAMP and Protein Kinase A
To examine the role of the adenylate cyclase, cAMP, and protein kinase A signaling pathway in the PACAP and VIP-induced progesterone response, the cells (5 x 104 cells/well) were incubated for 2 h with forskolin (10-5 M) and dibuturyl cAMP (10-3 M; Sigma) or the protein kinase A inhibitor H89 (0.5 x 10-6 M and 5 x 10-6 M; Calbiochem, San Diego, CA), alone or in combination with approximately half-maximal doses of PACAP 38 (5 x 10-9 M) or VIP (5 x 10-8 M). H89 was added 15 min before the peptides. Culture medium was obtained after centrifugation and analyzed for progesterone. Data (in nanomoles per liter) are presented as the mean ± SEM from five different experiments, each performed in duplicate.
Statistical Analysis
Statistical analysis was performed with ANOVA followed by Dunnett test for multiple comparisons or with the paired t-test for paired comparisons. Curve fittings and calculations of IC50 and EC50 values were performed with GraphPad Prism 2.01 using nonlinear regression. P values < 0.05 were considered significant.
RESULTS
RT-PCR of PACAP receptor mRNA
Reverse-transcription polymerase chain reaction analysis revealed the presence of PAC1 mRNA splice variants (Fig. 1A). Two bands, of ~300 and ~390 bp, were identified using primers P1/P4 (Fig. 1A, lane 2). The ~300-bp band corresponds to the expected size of the basic variant. The ~390-bp band corresponds to the hop 1/hop 2 variants, as confirmed by the absence of bands using primers P2/P4 and of a ~260-bp band using primers P3/P4. The hop 1 and hop 2 variants differ in size by only 3 bp and are thus indistinguishable.
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A distinct band of ~390 bp was observed with the VPAC2 specific primers corresponding to the expected size. No band was observed using the VPAC1 specific primers (Fig. 1B). All water controls were negative.
Receptor Ligand-Binding Assay
125I-Labeled PACAP 27 binding to granulosa/lutein cells was dose-dependently displaced by unlabeled PACAP 38 with an IC50 value of 0.2 nM. Unlabeled VIP also dose-dependently displaced 125I-labeled PACAP 27 binding, and significant displacement was observed at 10-9 M and higher concentrations. At maximum concentrations of unlabeled VIP (10-6 M), only approximately 50% displacement of maximum specific binding was observed (Fig. 2A). The maximum specific 125I-labeled PACAP 27 binding activity constituted 9.3% ± 2.3% of the total tracer activity.
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125I-Labeled VIP binding to the cells was dose dependently and identically displaced by both PACAP 38 and VIP with IC50 values of 10 and 11 nM, respectively (Fig. 2B).
The maximum specific 125I-labeled VIP binding activity constituted 2.9% ± 0.2% of the total tracer activity.
Receptor Autoradiography
Binding of both 125I-labeled PACAP 27 (Fig. 3, A and C) and 125I-labeled VIP (not shown) was observed on granulosa/lutein cells, cumulus cells, and oocytes. As demonstrated in Figure 3, A and C, intense labeling, significantly above the background labeling, was a constant observation on 10–15% of the somatic cells and 20–25% of the oocytes. When incubations were performed in the presence of PACAP 38 (1 µM; Fig. 3, B and D) or VIP (1 µM; not shown), this intense labeling was abolished.
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cAMP and Progesterone Accumulation: Effects of PACAP 38 and VIP
PACAP 38 and VIP dose-dependently stimulated cAMP accumulation with EC50 values of 0.2 nM and 94 nM, respectively (Fig. 4A).
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The two peptides also induced a dose-dependent progesterone accumulation with EC50 values of 7.8 and 470 nM, respectively (Fig. 4B).
Progesterone Accumulation: Influence of cAMP and Protein Kinase A
Progesterone accumulation was significantly (P < 0.05) stimulated by 10-5 M forskolin (16.4 ± 8.3 nM) and 10-3 M dibutyryl cAMP (4.9 ± 0.6 nM), compared with control values (2.9 ± 0.1 nM).
Basal progesterone accumulation (control) was reduced to 1.9 ± 0.4 nM by 0.5 µM H89 and was significantly inhibited to 1.3 ± 0.2 nM by 5 µM H89 (P < 0.05).
Progesterone accumulation induced by 5 nM PACAP 38 (11.0 ± 3.7 nM) was significantly and dose-dependently inhibited to 7.7 ± 2.7 nM by 0.5 µM H89 and to 2.6 ± 0.3 nM by 5 µM H89 (P < 0.05 and 0.01, respectively). Progesterone accumulation after 50 nM VIP (19.1 ± 8.1 nM) was reduced to 15.5 ± 7.5 nM by 0.5 µM H89 and was significantly inhibited to 2.3 ± 0.4 nM by 5 µM H89 (P < 0.05). See Figure 5 for illustration of this entire section.
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DISCUSSION
Progesterone is an important and possibly obligatory intraovarian regulator of ovulation and luteinization, mediating its effect locally via nuclear progesterone receptors transiently expressed in preovulatory follicular cells [1, 2, 26, 27]. The LH surge undoubtedly initiates the rapid preovulatory rise in progesterone production. However, immunoneutralization of endogenous PACAP released from preovulatory follicular cells partially inhibits acute basal progesterone accumulation [14], suggesting that PACAP mediates some of this effect during later stages of the preovulatory period. In this study, we focused on the receptors and the signaling pathway involved in the effect of PACAP on acute progesterone production from such cells, and a number of methodological approaches were undertaken to demonstrate and characterize the presence of two of the three different PACAP-binding receptors on granulosa/lutein cells as well as on oocytes.
PACAP exists in two biologically active forms, PACAP 27 [28] and PACAP 38 [3], of which PACAP 38 is the predominant form in most tissues, including the ovary [4, 24, 29–31]. On the basis of binding affinities, PACAP receptors are divided into two types [4, 16]: PACAP type I receptors, which bind PACAP 38 and PACAP 27 with much higher affinity (1000 times or more) than the closely related peptide VIP, and PACAP type II receptors, which bind PACAP 38, PACAP 27, and VIP with equal affinity. One of the cloned receptors, named PAC1 [16], is a PACAP type I receptor [17]. The two other receptors, named VPAC1 [18] and VPAC2 [19], bind PACAP 38, PACAP 27, and VIP with nearly equal affinity and thus are PACAP type II receptors.
Employing RT-PCR with specific primers for the different splice variants of PAC1, we demonstrated mRNA from the basic and the hop variants that seem to be the predominant forms in most tissues [23, 32]. Radioligand-binding studies with 125I-labeled PACAP 27 supported the findings by demonstrating the presence of functional PACAP type I receptor-binding sites. Binding of 125I-labeled PACAP 27 was potently displaced by PACAP 38, in contrast to a limited displacement by VIP reaching only approximately 50% of maximum binding. This pattern is characteristic for the PACAP specific PACAP type I receptor. However, significant displacement of 125I-labeled PACAP 27 was observed already at nanomolar concentrations of VIP, suggesting the simultaneous presence of a high-affinity VIP-binding site as well. Accordingly, PACAP type II-binding sites were demonstrated by the equipotent displacement of 125I-labeled VIP binding by both PACAP 38 and VIP. The IC50 values were within the reported range of the cloned VPAC2 receptor [16, 19], and in agreement with a previous study [20], the mRNA from this receptor subtype was demonstrated by RT-PCR, whereas no VPAC1 mRNA was observed.
By autoradiography, both 125I-labeled PACAP 27 and 125I-labeled VIP binding were observed on granulosa/lutein cells and on oocytes. Although cells with relatively low expression of receptors may not be identified with this method, intense labeling was observed on a minor proportion of the cells. This could indicate the presence of a subpopulation of follicular cells with high expression of PACAP receptors, a notion supported by the previous demonstration that VIP stimulates steroidogenesis in a subpopulation of ovarian granulosa cells that are unresponsive to FSH [33]. However, because the cells were pooled from different animals, the finding could also reflect interindividual or interfollicular differences due to, or caused by, regulation of the receptors. In situ hybridization on ovarian sections collected at short intervals during the preovulatory period and double-labeling experiments for the receptors, PACAP, and the cholesterol side-chain cleavage cytochrome P450 could be helpful in determining the relevant cell population in the preovulatory follicles as well as any regulatory changes.
Both PAC1 and VPAC2 are coupled to adenylate cyclase [4, 16, 17, 19]. In accordance, PACAP 38, supposedly interacting with both receptors, and VIP, interacting predominantly with the VPAC2 receptor, stimulated cAMP formation. PACAP 38 showed intimate concordance between receptor binding and second-messenger activation, in contrast to the approximate 10-fold discordance observed with VIP. A similar pattern has been described in other cell types displaying simultaneous expression of PAC1 and VPAC2 [32]. Because the EC50 of cAMP production via the secretin receptor, another member of this receptor family, depends on the number of secretin receptors expressed on a cell [34], it was suggested that the difference could indicate the predominant presence of PAC1 receptors in the cell population [32]. In the present study, a similar notion is supported by the observed threefold-higher maximum specific-binding activity of 125I-labeled PACAP 27, compared with 125I-labeled VIP. Alternatively the PAC1 receptor is more efficiently coupled to adenylate cyclase or may compete more effectively for available G-proteins than the VPAC2 receptor. In any case, although small amounts of VIP mRNA [35] and VIP immunoreactivity has been demonstrated in rat preovulatory ovaries [36] and in human preovulatory follicular fluid [37], the present findings suggest that PACAP is the physiologically important of the two peptides in preovulatory follicles.
Following the LH surge, the granulosa cells of preovulatory follicles undergo the differentiation process known as luteinization. The genetic reprogramming that underlies this process is completed during the preovulatory period and results in cessation of cell growth, an increase in cell volume, and appearance of lipid droplets. Furthermore, the cholesterol side-chain cleavage cytochrome P450 enzyme becomes constitutively elevated, and progesterone production changes from being a cAMP-dependent to a cAMP-independent process [2, 27, 38]. Preovulatory follicular cells are intermediaries in this process (hence the name "granulosa/lutein cells"), and in these cells, acute progesterone production has been associated with multiple intracellular pathways [38]. In this study, we demonstrated that the effects of PACAP 38 and VIP on acute progesterone production in granulosa/lutein cells are coupled to cAMP and the protein kinase A pathway. Both the adenylate cyclase activator forskolin and the cAMP analogue dibuturyl cAMP stimulated acute progesterone accumulation, and the progesterone-accumulating effects of PACAP 38 and VIP were inhibited by the protein kinase A inhibitor H89. However, additional signaling pathways may be involved because the two receptors activate multiple intracellular pathways [17, 19], and both PACAP and VIP are known to stimulate steroidogenesis by cAMP-independent pathways in other steroidogenic cells [39–42].
Interestingly, 125I-labeled PACAP 27 and 125I-labeled VIP binding were also observed on oocytes. This agrees at least in part with the recent demonstration that PACAP (but not VIP) stimulates cAMP accumulation and inhibits spontaneous maturation in denuded preovulatory oocytes [10]. Mammalian oocytes are arrested in the dictyate stage until shortly before ovulation. This inhibition is associated with high levels of cAMP inside the oocyte [43], and activation of PACAP receptors located on the oocyte therefore could be involved. Resumption of meiosis takes place in preovulatory follicles as a result of the midcyclic gonadotropin surge. It is a complex process involving increasing concentrations of cAMP in surrounding follicular cells and decreasing cAMP concentrations in the oocyte [43]. Both PACAP and VIP stimulate resumption of meiosis in follicle-enclosed oocytes in vitro [10, 44], and therefore, depending on the cellular localization or putative regulatory mechanisms, PACAP receptors could be involved in different aspects of oocyte maturation.
In summary, we have demonstrated that two of the three known PACAP receptors are present on preovulatory granulosa/lutein cells and on oocytes. The receptors mediate the acute effect of PACAP on progesterone production, and the process is cAMP dependent. The data provide further evidence to establish PACAP as an autocrine or paracrine regulator of LH-induced acute progesterone production in rat preovulatory follicles.
FOOTNOTES
First decision: 2 November 2000.
1 The study was supported by the Danish Biotechnology Center for Cellular Communication. 
2 Correspondence: Søren Gräs, Department of Clinical Biochemistry, University of Copenhagen, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark. FAX: 45 35 31 39 55; s.gras{at}dadlnet.dk
Accepted: February 28, 2000.
Received: October 5, 1999.
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