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Hormone Research Center (H.-S.C., B.-J.L., W.-S.C., S.-Y.C.) and Department of Biology (J.L., H.-J.P., H.-B.K.), Chonnam National University, Kwangju 500–757, Republic of Korea; and US-Japan Biomedical Research Laboratories (A.A.), Tulane University Hebert Center, Belle Chasse, Louisiana 70037
Address all correspondence and requests for reprints to: Sang-Young Chun, Hormone Research Center, Chonnam National University, Kwangju 500–757, Korea. E-mail: sychun{at}orion.chonnam.ac.kr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-amanitin
abolished LH-induced PACAP transcripts, indicating that new protein
synthesis and transcription are necessary. Treatment with
MDL-12,330A, an inhibitor of adenylate cyclase, inhibited
LH-induced PACAP messenger RNA, and forskolin mimicked the LH action,
implying the role of adenylate cyclase activation. In contrast,
treatment with chelerythrine, an inhibitor of protein kinase C, and
2-O-tetradecanol-phorbol-13-acetate had no
effect. We further tested the role of PACAP in follicle apoptosis using
apoptotic DNA fragmentation analysis. Treatment with PACAP-38
suppressed follicle apoptosis in a dose-dependent manner. Moreover, the
LH suppression of follicle apoptosis was partially blocked by
cotreatment with PACAP-38 antagonist, indicating mediation by
endogenous PACAP-38. These results suggest that PACAP, transiently
induced by the gonadotropin surge, could be a local regulator of a
number of events and may act as a follicle survival factor during the
periovulatory period. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies of tissue distribution have shown that both PACAP and its receptors are present not only in the central nervous system but also in a variety of peripheral tissues, such as the testis, adrenal, and ovary (9, 10). In the ovary, the presence of immunoreactive PACAP-38 as a predominant form has been demonstrated by RIA in whole-tissue extract in humans (11) and rats (12). Binding sites for PACAP (13) and the presence of PACAPR-I messenger RNA (mRNA) (14) have also been demonstrated in cyclic rat ovaries. Recent studies have shown that PACAP stimulates steroidogenesis and cAMP accumulation more potently than VIP in cultured rat granulosa cells (15, 16). Furthermore, PACAP regulates meiotic maturation, mimicking the action of LH in rat oocytes (17), suggesting a role of PACAP in the regulation of ovarian function as a new local factor. Of relevance to the present work, it has been shown that PACAP mRNA is found primarily in granulosa cells of preovulatory follicles and some theca/interstitial cells and regulated in vivo by gonadotropins in the rat ovary (18).
In the present study, we elucidated more detailed time-dependent induction of PACAP mRNA by gonadotropins both in ovaries of PMSG/human (h)CG-treated immature rats and in cultured preovulatory follicles. We found that gonadotropins could induce a transient expression of PACAP transcripts, which was maximal at 6 h after stimulation in granulosa cells of preovulatory follicles, possibly by activating adenylate cyclase. We also demonstrated the role of PACAP in the suppression of follicle apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-amanitin, forskolin, and
2-O-tetradecanol-phorbol-13-acetate (TPA) were purchased
from Sigma Chemical Co. (St. Louis, MO). PACAP-38 and
PACAP (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), an antagonist of PACAP-38, were obtained from Bachem
(Torrance, CA). MDL-12,330A and chelerythrine (CE) chloride were
purchased from Calbiochem (San Diego, CA). The
progesterone antiserum was donated by Dr. Yong-Dal Yoon
(Hanyang University, Seoul, Korea).
Animals
Immature (26-day-old; BW, 60–65 g) female Sprague Dawley rats
(Daehan Laboratories, Chungbuk, Korea) were injected sc with 10 IU PMSG
to induce multiple follicle growth. At 48–52 h later, the animals were
killed by cervical dislocation, and the ovaries were removed for
follicle dissection. In pilot experiments, these rats responded
optimally to an ovulatory dose (10 IU) of hCG at 2 days after 10-IU
PMSG treatment, based on their ovulation rate (34.4 ± 5.5 ova per
rat; n = 9). Some rats received a single ip injection of 10 IU hCG
to induce ovulation, and ovaries were obtained at different time
intervals for Northern blot and in situ hybridization
analysis.
Follicle culture
Preovulatory follicles (>800 µm in diameter) were isolated
from ovaries collected at 48–52 h after PMSG injection, and follicle
culture was performed as previously described (19, 20). For follicle
apoptosis studies, two follicles were cultured in glass vials
containing 400 µl Eagle’s MEM (Gibco, Grand Island, NY) supplemented
with penicillin, streptomycin, L-glutamine, and 0.1% BSA
(wt/vol, Fraction V, Sigma Chemical Co., St. Loius, MO) in
the absence or presence of different hormones. In some cultures,
fifteen to twenty follicles were incubated in 800 µl of medium for
studies of PACAP mRNA expression. Cultures were maintained for up to
24 h at 37 C under 5% CO2-95% O2. At the
end of incubation, follicles were snap-frozen for DNA and RNA isolation
or were fixed for in situ hybridization analysis. The media
were collected for the determination of progesterone
levels by RIA.
Northern blot analysis
Total RNA from ovaries or cultured follicles was isolated using
Tri Reagent solution (Molecular Research Center, Inc.,
Cincinnati, OH). Twenty micrograms of total RNA were fractionated by
electrophoresis on a 1.2% agarose gel containing formaldehyde and were
transferred to nylon membranes by capillary blotting with 20 x
sodium citrate-sodium chloride (SSC). After a UV cross-linking and
prehybridization, membranes were hybridized overnight at 65 C in a
solution containing 50% formamide, 5 x SSC, 1.6 x
Denhardt’s solution, 1 mM EDTA, 250 µg/ml denatured
herring sperm DNA, 500 µg/ml yeast transfer RNA, and a total of
2–4 x 106 cpm of a 32P-labeled rat PACAP
complementary DNA (cDNA) probe (21). After hybridization, membranes
were washed twice for 5 min at room temperature in 2 x SSC and
0.1% SDS, followed by 1 h at 65 C in 0.5 x SSC and 0.1%
SDS. Membranes were then exposed using Kodak RX films (Eastman Kodak Co., Rochester, NY) for 2–5 days for ovarian samples and
for 2–12 h for cultured follicular samples at -80 C. For
normalization of data, blots were stripped by boiling in 0.1 x
SSC and 0.5% SDS twice for 20 min before reprobing with a cDNA probe
for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) (22).
In situ hybridization analysis
Ovaries or cultured follicles were fixed at 4 C for 6 h in
4% paraformaldehyde in PBS, followed by immersion in 0.5 M
sucrose in PBS overnight. Cryostat sections (14-µm thick) were
mounted on poly-L-lysine (Sigma Chemical Co.)-coated microscope slides, fixed in 4%
paraformaldehyde in PBS, and stored at -80 C until analyzed. The
hybridization procedure was essentially the same as previously
described (23). In brief, sections were pretreated serially with 0.2
M HCl, 2 x SSC, pronase (0.125 mg/ml), 4%
paraformaldehyde, and acetic anhydride in triethanolamine.
Hybridization was carried out at 52–55 C overnight in the mixture
containing 35S-labeled rat PACAP cRNA probe
(108 cpm/ml), 50% formamide, 0.3 M NaCl, 10
mM Tris-HCl, 5 mM EDTA, 1 x Denhardt’s
solution, 10% dextran sulfate, 1 µg/ml carrier transfer RNA, and 10
mM dithiothreitol. Posthybridization washing was performed
under stringent conditions that included ribonuclease A (25 µg/ml)
treatment at 37 C for 30 min and a final stringency of 0.1 x SSC.
Slides were dipped into NTB-2 emulsion (Eastman Kodak Co.)
and exposed at 4 C until being developed after 2 weeks. The slides were
stained with hematoxylin and eosin and were examined under the light
microscope with bright- and dark-field illumination.
DNA fragmentation analysis
Cellular DNA was extracted from follicles and analyzed as
previously described (20, 24). Aliquots of 0.5 µg DNA from each
culture were labeled at 3'-ends with [32P]dideoxy-ATP
(3,000 Ci/mmol; Amersham, Arlington Heights, IL) using terminal
transferase (Boehringer Mannheim, Indianapolis, IN). Equal amounts (200
ng/lane) of labeled DNA samples for each treatment group were
fractionated through 2% agarose gels. Gels were dried in a slab gel
dryer and exposed using Kodak x-ray films (Eastman Kodak Co.) at -80 251 C. After autoradiography, portions of each lane
corresponding to DNA less than 15 kb (kb) were isolated and counted in
a ß-counter for quantitation of the degree of apoptotic DNA
fragmentation. All data are expressed as per cent of the control group
at 24 h of culture.
RIA
Concentrations of progesterone in culture media
were determined by RIA, as previously described (25), and expressed as
ng/ml.
Data analysis
Statistical differences were assessed by one-way ANOVA, followed
by Student’s t test; and P < 0.05 was
considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The levels of mRNA
were transiently induced by hCG administration, reaching a maximum
induction 6 h after hCG treatment. The most intense band had a
size of 1.2 kb. The 2.4- and 3.0-kb bands corresponded in size to those
observed in the hypothalamus. PACAP specific bands were not detected at
any time points in postnatal, estrogen-treated, or PMSG-primed ovaries
(data not shown).
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, B and E), but not
growing follicles (Fig. 2, B and H). Under higher magnification, it was
observed that the parietal granulosa cells expressed higher levels of
PACAP mRNA than the expanded mature cumulus cells (Fig. 2F). Specific
signals for PACAP transcripts were also detected in a few scattered
solitary interstitial cells and in some theca cells, regardless of
follicle size (Fig. 2; B, H, and I). Adjacent sections hybridized with
the sense cRNA probe did not show any hybridization signal (Fig. 2C).
Specific signals were not detected at any time points in PMSG- or
estrogen-treated ovaries (data not shown).
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). The
effect of LH on PACAP mRNA stimulation delayed, relative to that of
FSH; levels of PACAP transcripts remained elevated at 12 h after
LH treatment (Fig. 3A), whereas they were barely detectable after FSH
treatment (Fig. 3B). PACAP transcripts were not detectable in follicles
before culture and 24 h after gonadotropin addition. As shown in
Fig. 4, both LH and FSH treatment for
6 h induced PACAP mRNA in a dose-responsive manner,
reaching saturating levels at 100 ng/ml.
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). The parietal granulosa cells had
higher signals than the expanded cumulus cells (Fig. 5C), comparable
with the expression seen in vivo 6 h after hCG
administration (Fig. 2F
). However, theca cells were devoid of specific
signals (Fig. 5C). No specific signal was detected in follicles
hybridized with sense probe (Fig. 5D).
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-amanitin on LH-induced PACAP mRNA
in cultured preovulatory follicles-amanitin. The protein synthesis inhibitor,
cycloheximide, blocked the LH induction of PACAP transcripts in a
dose-dependent manner, a marked decrease at 1 µg/ml (Fig. 6A). Inclusion of -amanitin, an
inhibitor of transcription, also blocked the LH induction of PACAP
transcripts, with a complete abolishment at 25 µg/ml (Fig. 6B).
Treatment of follicles with cycloheximide or -amanitin alone had no
effect.
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). In contrast, the addition of CE
did not affect the LH action on stimulation of PACAP mRNA. Moreover,
forskolin treatment stimulated the induction of PACAP transcripts,
whereas treatment with the phorbol ester TPA had no effect. Follicles
incubated with MDL or CE alone did not show any PACAP transcripts.
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). Incubation of follicles with
PACAP-38 caused a dose-related reduction in the extent of apoptotic DNA
cleavage (52.2% of 24-h controls at 10-6 M).
Furthermore, cotreatment with PACAP-38 specific antagonist (P38-A)
prevented the suppressive effect of PACAP-38. Because LH increased
PACAP transcript levels in preovulatory follicles (Fig. 3), we
determined whether the suppressive effect of LH on follicle apoptosis
is mediated via the action of endogenous PACAP. As shown in Fig. 8B, LH-induced suppression of follicle apoptosis was partially inhibited
(47.1%) by cotreatment with PACAP-38 antagonist (10-5
M). Treatment with PACAP-38 antagonist alone had no effect
on follicle apoptosis.
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, levels of progesterone
were increased by LH (8-fold). Treatment with PACAP-38 caused a
dose-dependent increase in progesterone production (4-fold
at 10-6 M), and this increase was partially
inhibited by the addition of PACAP-38 antagonist (10-5
M).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although immunoreactive PACAP, mainly in the form of PACAP-38, has been reported to be present in the ovary (11, 12), it has only recently been demonstrated by Gräs et al. (18) that gonadotropins can induce a transient expression of PACAP mRNA in the rat ovary. In that report, hCG administration to PMSG-primed immature rats stimulated PACAP mRNA expression in granulosa cells of preovulatory follicles and in some theca/interstitial cells, with a maximum induction around 12 h after stimulation; whereas PMSG administration stimulated PACAP mRNA expression only in some theca/interstitial cells (18). In the present study, however, PMSG administration to immature rats was unable to stimulate PACAP mRNA expression at any time points examined by Northern blot and in situ hybridization analysis (data not shown). Furthermore, hCG injection induced a transient expression of PACAP mRNA, reaching a maximum at 6 h and being barely detectable at 12 h after stimulation. At present, we cannot explain the reason for the difference in PMSG effect and timing of hCG-induced PACAP mRNA induction between our present findings and work of Gräs et al. It may be because of the difference in hormone responsiveness derived from the difference in maturity of the test animals (26-day-old Sprague Dawley vs. 20- to 23-day-old Wistar rats).
To study the mechanism of stimulatory action of gonadotropins on PACAP mRNA expression, we have employed in vitro culture of preovulatory follicles obtained from PMSG-primed rats. Treatment with exogenous LH or FSH in cultured follicles could induce a transient expression of PACAP mRNA in a dose-dependent manner, mimicking the expression pattern of PACAP mRNA after hCG injection in vivo. Northern blot analysis revealed three different PACAP transcripts. One transcript, 1.2 kb in size, has been reported to be expressed in the rat testis (27). The other two transcripts (2.4 and 3.0 kb in size) were similar to those observed in nervous tissue (28). The major cell type expressing PACAP mRNA in cultured preovulatory follicles was granulosa cells. However, unlike in vivo hCG-treated ovaries, theca cells were devoid of PACAP-specific signal in response to LH. Furthermore, PACAP mRNA remained elevated for up to 12 h in preovulatory follicles cultured in vitro with LH, compared with ovaries treated with hCG in vivo, indicating that the mechanism mediating the rapid decline in PACAP mRNA after hCG treatment in vivo is reduced in vitro after LH treatment. A similar observation has been reported in the induction of PG endoperoxide synthase (29). Accordingly, one possible explanation for the difference is that cAMP production seems to be sustained longer in vitro after LH treatment (30).
It has been indicated that low concentrations of LH preferentially
activate adenylyl cyclase, whereas higher concentrations of the hormone
also increase intracellular calcium and activate protein kinase C (PKC)
(31, 32). The present findings, showing the inhibition of LH action on
the induction of PACAP mRNA by adenylyl cyclase inhibitor, but not by
PKC inhibitor, provide evidence to hypothesize a primary role for cAMP
in PACAP mRNA expression in preovulatory follicles. Indeed, it has been
reported that the 5' flanking region of the hPACAP gene contains
sequence motif homologous to cAMP response element (33). Furthermore,
the induction of PACAP mRNA by LH in vitro was blocked by
either cycloheximide or -amanitin, indicating that translational and
transcriptional events are involved in cAMP-mediated induction of PACAP
mRNA. Similar observations have been reported in the transient
appearance of progesterone receptor (34) and PG
endoperoxide synthase mRNAs (29) in preovulatory follicles after LH/hCG
stimulation.
The preovulatory surge of gonadotropins is obligatory, to trigger the ovulatory process (including follicle rupture, oocyte maturation, and luteinization) (35). The mature follicles not exposed to the ovulatory signal undergo apoptotic degeneration (36, 37), and recent studies demonstrate that insulin-like growth factors I and Il-1ß play a role in mediating the gonadotropic action on the suppression of follicle apoptosis (19, 20). Thus, our observation, showing the transient stimulation of PACAP mRNA during LH/hCG-induced ovulation, prompted us to examine the role of PACAP in follicle apoptosis. The present study demonstrated that treatment with PACAP-38 dose-dependently suppresses apoptotic DNA fragmentation in the follicles, indicating that PACAP-38 is capable of protecting preovulatory follicles from apoptotic death. Furthermore, the suppressive effect of LH was partially reversed by the addition of PACAP-38 antagonist, suggesting that part of the action of LH is also mediated by endogenously produced PACAP-38. PACAP-38 has been shown to inhibit apoptosis in neuronal cells by activating the mitogen-activated protein kinase through a cAMP-dependent pathway (38). Because PACAP is known to stimulate cAMP accumulation in ovarian granulosa cells (15) and the suppression of follicle apoptosis is mediated by cAMP pathway (39), PACAP-38 might inhibit preovulatory follicle apoptosis by increasing cAMP levels in granulosa cells.
PACAP can interact with specific receptors. The type I receptor binds preferentially to PACAP, and the type II binds PACAP and VIP with similar affinity (3). The present observation, demonstrating a dose-dependent increase in progesterone production by PACAP-38, implicates the presence of PACAP receptors in preovulatory follicles. Indeed, PACAP has been shown to be more potent than VIP in the stimulation of cAMP and steroidogenesis in cultured granulosa cells from estrogen-treated immature rats (15, 16), suggesting the presence of PACAP type I receptor. In a recent study, PACAP has been shown to accelerate oocyte maturation in follicle-enclosed, cumulus-enclosed, and denuded oocytes, whereas VIP is effective only in follicle-enclosed oocytes (17). The data from that study imply that different subtypes of PACAP receptors are present in the different ovarian compartments.
The LH surge is known to induce expression of several genes associated
with follicle rupture and luteinization (40). The rapid, but transient,
expression of the progesterone receptor (34), tissue
plasminogen activator (41), Il-1ß (42), and PG endoperoxide synthase
(29) genes seems to be temporally and functionally associated with the
process of follicle rupture. In contrast, the biochemical changes that
are associated with luteinization include the sustained induction of
P450scc (43), the transient increase in progesterone
receptor (34), aromatase (44), and 17-hydroxylase (45). Whether the
dramatic and transient expression of PACAP, after LH/hCG treatment,
plays a critical role in the ovulatory process remains to be
elucidated.
In summary, gonadotropin induction of PACAP mRNA in granulosa cells of preovulatory follicles is mediated by cAMP, involving translational and transcriptional controls. The transient appearance of PACAP plays a role in the suppression of follicle apoptosis and may involve novel intracellular factors and the regulatory processes during the periovulatory period.
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Acknowledgments |
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Footnotes |
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Received May 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene promoter. Mol Endocrinol 9:1356–1366[Abstract]
, cytochrome P450scc and
3-hydroxy-3-methyl CoA reductase in developing rat ovarian follicles
and corpora lutea: correlation with theca cell steroidogenesis. Biol
Reprod 37:211–223[Abstract]
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