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Department of Clinical Biochemistry, University of Copenhagen, Bispebjerg Hospital, Copenhagen, Denmark
Address all correspondence and requests for reprints to: Søren Gräs, Department of Clinical Biochemistry, University of Copenhagen, Bispebjerg Hospital, Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pituitary adenylate cyclase-activating polypeptide (PACAP), the latest member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of peptides (6, 7), has the potential of being a local regulator of ovarian physiology (8, 9, 10, 11, 12, 13, 14, 15) and periovulatory progesterone production, in particular (16). PACAP was originally isolated from hypothalamus and exists in two biologically active forms, PACAP 27 and PACAP 38, of which PACAP 38 is the dominating form in tissue (6, 7, 17). PACAP is considered a neuropeptide, based on its presence in distinct areas of the CNS and in neuronal elements from a number of peripheral organs (7, 18, 19, 20, 21, 22, 23, 24), including the ovary (16, 25, 26). However, PACAP is also expressed in nonneuronal cells, as PACAP immunoreactivity (PACAP-IR) and PACAP mRNA have been shown in spermatogenic cells from the rat testis (27, 28, 29) and in steroidogenic cells from the rat ovary (16).
PACAP expression in ovarian steroidogenic cells is transient and confined to the periovulatory period (16). The spatiotemporal expression of the peptide coincides with high expression of elements from the periovulatory cascade involved in progesterone production, such as the LH-receptor (2), the steroidogenic acute regulatory protein (StAR), the rate-limiting step in steroidogenesis (5), and the cholesterol side chain cleavage cytochrome P450 (P450scc), responsible for the first reaction in progesterone biosynthesis (5, 30). Furthermore, PACAP is a potent stimulator of cAMP formation and is known to stimulate cAMP and steroidogenesis in ovarian cells (8, 10, 11, 12, 13, 15), an action it shares with the gonadotropins. Thus, several lines of evidence suggest that PACAP could be an auto- or paracrine regulator of periovulatory progesterone production. To test this hypothesis, we established an in vitro system using ovarian granulosa/lutein cells primed in vivo with PMSG and human CG (hCG), to mimic the characteristic influence of the gonadotropins; and examined: 1) whether granulosa/lutein cells accumulated and secreted immunoreactive PACAP; 2) whether exogenous PACAP induced progesterone accumulation in the culture medium; and 3) whether immunoneutralization of endogenously secreted PACAP with a specific PACAP antiserum influenced acute progesterone accumulation and subsequent functional and morphological signs of luteinization in the cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells
Granulosa/lutein cells were harvested 4–6 h after the hCG
injection. The animals were decapitated, and the ovaries were removed
aseptically, freed from adherent tissue, and transferred to a Petri
dish with culture medium (McCoys 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 centrifugated 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.
Culture conditions
Short-term incubation. Short-term incubation (1–24 h) was
used to study the presence of endogenous PACAP and the acute effects of
both endogenous and exogenous PACAP. This period corresponds, more or
less, to the periovulatory period during which PACAP expression is
observed in granulosa/lutein cells under in vivo conditions
(16). In experiments examining the presence of and effects of
endogenous PACAP, cells were diluted to 5 x 105
cells/ml in McCoys 5A culture medium supplemented with penicillin,
streptomycin, and testosterone (10-7 M;
Sigma Chemical Co.; final ethanol concentration, 0.01%
vol/vol). In experiments examining the effects of exogenous PACAP38,
the cells were diluted to 2.5 x 105 cells/ml to
reduce the possible influence of endogenous PACAP. Aliquots of 200 µl
were placed in 96 multiwell dishes (Nunc, Copenhagen, Denmark), and
incubation was performed at 37 C in 5% CO2-95% air. Under
these conditions, the cells do not adhere to the wells. The 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 were
analyzed for progesterone or PACAP 38. The cell pellets were either
stored at -20 C for later PACAP 38 measurement or were processed for
immunocytochemistry, as described below.
Long-term incubation. Long-term incubation (1–10 days) was used to study the effects of endogenous PACAP on the functional and morphological changes associated with luteinization [increased progesterone production, cell hypertrophia, and lipid droplet accumulation (2, 3)]. During the first 24 h, incubation conditions were as described above. The cell suspensions were then aspirated and centrifuged at 250 x g for 5 min. The supernatants were stored at -20 C and later analyzed for progesterone. The cells were resuspended (105 cells/ml) in fresh culture medium supplemented with FCS 1%, and 0.2-ml aliqouts were seeded in 96 multiwell dishes and incubated for 9 additional days. Under these conditions, a monolayer of cells is formed after 2–4 days in culture, as described earlier (3).
Demonstration of PACAP
PACAP 38 RIA. The concentration of PACAP 38 in extracted
cells (105 cells/well) was analyzed after incubation for
0.5, 1, 8, and 24 h using a previously described RIA specific for
PACAP 38 (18). The concentration of PACAP in the corresponding culture
medium samples was measured using a modified RIA buffer. Standards were
dissolved in a buffer that was made by reconstitution of freeze-dried
fresh McCoys 5A medium in 3 vol normal assay buffer. Similarly, the
unextracted medium samples were freeze-dried and reconstituted in 3 vol
normal assay buffer before analysis in duplicate. The intra- and
interassay coefficients of variation of the PACAP 38 assay were 3.0%
and 7.2%, respectively. The detection limit of the assay in modified
buffer was 15 pmol/liter. Six different experiments were performed.
Each experiment was done in triplicate, and cells and media from each
experiment were pooled before analysis.
PACAP immunocytochemistry. Immunocytochemical visualization of PACAP was carried out, as described previously, using a monoclonal PACAP antibody (MabJHH1), which detects both PACAP 27 and PACAP 38 (16, 18, 26). The incubations were terminated at 0.5, 1, 3, 6, 8, and 24 h. After centrifugation, the cell pellets (105 cells/well) were resuspended in 50 µl Stefanini fixative and incubated for 1 h at room temperature. Drops of 20 µl were placed on a glass slide and were allowed to dry before the immunocytochemical processing.
Effect of exogenous PACAP 38 on progesterone accumulation. A time course was performed using PACAP 38 (Peninsula Laboratories, Inc.), diluted in culture medium to a final concentration of 10-7 M. The cells (5 x 104 cells/well) were incubated 0.5, 1, 2, 3, 6, and 8 h, with the peptide or culture medium as controls. Three different experiments were performed, each made in duplicate. Maximum response was observed at 2 h. At this time point, a dose-response study was performed with increasing concentrations of PACAP 38, diluted in culture medium (final concentrations 10-10–10-6 M). Six different experiments were performed, each made in duplicate. The effect of LH/hCG alone or in combination with PACAP 38 was examined using approximately half-maximal-effective doses of hCG (1 ng/ml) and PACAP 38 (5 x 10-9 M). Five different experiments were performed, each made in duplicate. Progesterone concentrations in the medium samples were assayed in duplicate with a commercial RIA kit (Orion Diagnostica, Espoo, Finland), as described earlier (31).
Immunoneutralization of exogenous PACAP. Neutralization of PACAP was performed using a specific PACAP antiserum (code No. 544C) raised in rabbit against synthetic PACAP 38 and characterized according to a previous report (18). The antiserum detects both PACAP 27 and PACAP 38 and does not cross-react with structurally related peptides. Nonimmune rabbit serum (Statens Seruminstitut, Copenhagen, Denmark) was used as control. The IgG fractions from both sera were isolated using a Protein G column (MAbtrap GII, Pharmacia4, Pharmacia Biotech, Uppsala, Sweden), according to the manufacturer’s instruction.
In initial experiments, the neutralizing capacity of the antiserum was assessed by the ability to inhibit the effect of added PACAP 38 on progesterone accumulation. The cells (105 cells/well) were incubated for 2 h with PACAP 38 (5 x 10-9 M) and increasing concentrations of the anti-PACAP IgG or nonimmune IgG (25, 50, and 100 µg/ml). Four different experiments were performed, each made in duplicate.
Immunoneutralization of the acute effects of endogenous PACAP. The cells (105 cells/well) were incubated for 1, 2, 3, 4, 6, and 8 h with anti-PACAP IgG (100 µg/ml) or nonimmune IgG (100 µg/ml), and culture media were collected for progesterone analysis. Five experiments were performed, each made in duplicate.
Immunoneutralization of the long-term effects of endogenous PACAP. The cells were incubated for 10 days. Culture medium was collected after 1, 3, 7, and 10 days and analyzed for progesterone. During the first 24 h, the cells were incubated with anti-PACAP IgG (100 µg/ml) or nonimmune IgG (100 µg/ml). Seven experiments were performed, each made in duplicate. The cells were examined daily in an inverted microscope.
Statistical analysis
Data are presented as mean ± SEM. Statistical
analysis was performed with ANOVA, followed by Student-Newman-Keuls
test or Dunnet’s test for multiple comparisons. In the experiments
examining the effects of endogenous PACAP, comparisons were performed
with the paired t test or the Wilcoxon rank-sum test.
P values < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In the cells, a PACAP-IR increase of
more than 5-fold was observed after 1 and 8 h, compared with 0.5
and 24 h (P < 0.002). In the culture medium,
PACAP-IR was detectable at 0.5, 1, and 8 h; and at 1 and 8 h,
the values were significantly higher than at 0.5 h
(P < 0.05).
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). From 1–8 h, an increasing number of
cells displayed PACAP immunostaining being maximal at 6 and 8 h
(Fig. 1B). At 24 h, no cells stained positive for PACAP.
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). At this time point, PACAP 38 induced
a dose-dependent rise in progesterone accumulation, with an estimated
EC50 value of approximately 7 nM (Fig. 2B).
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. Addition of both hCG and PACAP 38
induced an effect that was approximately the sum of their individual
effects.
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).
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).
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). After 4–10 days, a substantial number of the
nonimmune IgG-treated cells developed a homogenous luteal phenotype
with hypertrophic cell bodies, numerous cytoplasmic lipid droplets, and
large nuclei with distinct nucleoli, as described earlier (2, 3)
(Fig. 5A). Cells incubated with
anti-PACAP IgG showed a more heterogenous appearance. A minor number of
the cells displayed luteal phenotypes. The rest were generally smaller
and had either irregular or elongated forms resembling
fibroblasts (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Granulosa/lutein cells from PMSG/hCG-treated immature ovaries were used, and the cells were harvested from preovulatory follicles 4–6 h after the hCG injection. At this time, a transient PACAP expression begins in the cells and subsequently lasts for approximately 12 h in vivo (16). During incubation, the isolated granulosa/lutein cells used in the present study also transiently expressed PACAP. The time of appearance of PACAP-immonoreactivity was found to differ slightly, depending on whether immunocytochemistry or RIA was used. This apparent discrepancy could be explained by differences in sensitivity and specificity between the two methods because two different antibodies were used. The reported variations in the time of expression of PACAP, between PMSG/hCG-treated immature rats (16), could also contribute to the finding. However, a transient accumulation of PACAP-IR in the cells was demonstrated by both methods, and our culture system thus seems to imitate in vivo conditions. In addition, PACAP 38 was secreted from the granulosa/lutein cells, as evidenced by the presence of PACAP 38-immunoreactivity in the culture medium. The measured levels were somewhat low, compared with the concentration of exogenous PACAP needed to stimulate progesterone accumulation. However, the measured levels of endogenous immunoreactive PACAP in the culture medium could be low because of proteolytic degradation of the peptide during incubation. Furthermore, it is likely that the concentration of endogenous PACAP in the vicinity of the cells is higher than the measured average values in culture medium, a notion supported by the tendency of the cells to cluster during short-term incubation. Finally, the presence of endogenous PACAP close to the surface of the cells might influence the response of exogenous PACAP by shifting the dose-response curve to the right.
Exogenous PACAP induced a rapid and dose-dependent accumulation of progesterone in the medium; and in combination with hCG, an additive effect was observed. These findings suggest that intracellular pathways, shared by both LH/hCG (2) and PACAP (7), could be activated. Which receptor(s) and pathway(s) are involved is currently under investigation in our laboratory. PACAP has previously been reported to stimulate steroidogenesis in cultured granulosa cells from diethylstilbestrol-treated (8, 10) or PMSG-treated (15) immature rats, steroidogenesis in ovarian tissue cultures from the crested newt (11), and progesterone secretion in human luteal cell cultures (12). Furthermore, a stimulatory influence on ovarian steroidogenesis is established for the related peptide VIP (32, 33, 34), which shares sequence homology with PACAP and is known to interact with common receptors (7). Various stimulatory protocols and a variety of different culture conditions have been used in the above mentioned studies. Our demonstration that PACAP induces progesterone accumulation in ovarian cells from the periovulatory period during which PACAP is expressed in vivo is new. Interestingly, the rapid and transient response is different from previously described PACAP effects on ovarian cells. High concentrations of both side chain cleavage cytochrome P450 and StAR during this particular period in granulosa/lutein cells, providing optimal conditions for rapid progesterone production (5, 30), could offer an explanation for the rapid response to PACAP. The equally rapid return of progesterone to unstimulated values suggests close regulation of the response and extensive conversion of progesterone to other steroid metabolites.
Blockade of endogenously released PACAP was performed by immunoneutralization with a specific PACAP antiserum, which caused a dose-dependent inhibition of progesterone accumulation induced by exogenous PACAP. During incubation for 8 h, addition of the antiserum to the cultures significantly reduced progesterone accumulation, compared with controls. The 8-h incubation period corresponds to the latter half of the preovulatory period, and our findings thus imply that endogenously released PACAP, under normal conditions, stimulates progesterone production/secretion during this period.
How PACAP specifically fits into the sequence of biochemical events that regulate progesterone production in the preovulatory period is still speculative. Under physiological conditions, the midcyclic LH surge is the obligatory signal for the rapid preovulatory rise in ovarian progesterone production. However, ovarian progesterone production remains elevated throughout the preovulatory period, despite a concurrent decline in gonadotropin values. This suggests the existence of local supplementary mechanisms. The induction of the PACAP gene and the subsequent release and biological activity of the peptide during the latter half of the preovulatory period might constitute such a putative mechanism.
Immunoneutralization of endogenously released PACAP, during the first 24 h of a 10-day incubation period, induced functional and morphological signs indicative of impaired luteinization, i.e. a significant reduction of progesterone accumulation throughout the incubation period and a characteristic heterogenous and more undifferentiated cell morphology, compared with controls. These findings are in accordance with a previous report that demonstrated that luteinization of LH-stimulated preovulatory granulosa cells is similarly affected in vitro if a progesterone antagonist is applicated during the initial 7 h of incubation (3). Furthermore, several lines of evidence suggest that preovulatory ovarian progesterone production and the simultaneous transient induction of the progesterone receptor in granulosa/lutein cells are closely associated with ovulation and subsequent luteinization. Both are inhibited by progesterone synthesis inhibitors, progesterone antiserum, or progesterone antagonists (1, 2, 3, 4); and both fail to occur in progesterone receptor negative mice, despite occurrence of mature preovulatory follicles and appropriate exposure to gonadotropins (35). It is therefore possible that the inhibitory effect on luteinization observed in our study could be explained by the initial reduction in progesterone accumulation from PACAP immunoneutralized cultures.
In summary, we have previously demonstrated that the PACAP gene is transiently expressed in preovulatory granulosa/lutein cells in vivo. In the present study, we have demonstrated that PACAP is produced and secreted from granulosa/lutein cells in vitro and that the cells respond to PACAP with a rapid and dose-dependent increase in progesterone accumulation. Immunoneutralization of endogenous PACAP significantly reduced acute progesterone accumulation and impaired subsequent luteinization, suggesting an important auto- or paracrine role for PACAP during LH-induced periovulatory progesterone production.
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Acknowledgments |
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Footnotes |
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Received September 30, 1998.
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)
and vehicle controls (•). Values are the mean ± SEM
from three different experiments, each made in duplicate. B, Dose
response from granulosa/lutein cells incubated for 2 h with
increasing concentrations of PACAP 38 (
