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Hormone Research Center (H.-J.P., L.W., J.-H.P., H.-B.K., S.-Y.C.) and Department of Biology (J.L.), 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}chonnam.chonnam.ac.kr
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biological effects of PACAP are mediated through PACAP binding to at least three types of G protein-coupled seven transmembrane PACAP receptors. Type I PACAP receptors (PACAPR) have high affinity only for PACAP and type II PACAP receptors have high affinity for both PACAP and VIP, which is also known as the VIP receptor (VIPR) (10, 11). Spengler et al. (12) have identified five splice variants of the rat PACAPR differing only in their predicted third intracellular domains. The short variant form is designated PACAPR with the longer variant forms designated as hip, hop1, hop2, or hip-hop1 isoforms of the PACAPR. The splicing of the PACAPR gene has been shown to be differentially regulated in adult tissues (12). It has also been demonstrated that both PACAP-27 and -38 stimulate adenylate cyclase, whereas only PACAP-38 stimulates phospholipase C with high potency due to the existence of splice variant in extracellular domain of PACAPR (13). A third type of receptor (PACAPR-3) has recently been cloned in mice which binds PACAP and VIP with equal affinity (14). This PACAPR-3 displays high affinity helodermin, but not secretin, binding, whereas VIPR binds helodermin and secretin with lower affinities than for PACAP/VIP (14).
The PACAPR is expressed not only in the central nervous system but also in various peripheral tissues such as adrenal (12), testis (15), and ovary (16, 17), whereas the VIPR is predominant in the lung, liver, and gastrointestinal tract (11). Studies on the functional role of PACAP in steroidogenesis and oocyte maturation have suggested the presence of PACAP receptors in the ovary (7, 8, 9, 18). Furthermore, binding sites for PACAP have been demonstrated in cyclic rat ovaries (19). Although recent studies using the techniques of RT and PCR have shown the expression of messenger RNA (mRNA) encoding PACAPR in ovarian cells (16, 17), cell-type specific localization and hormonal regulation of PACAPR in the ovary have not been reported. The present study was therefore designed to investigate the tissue localization and gonadotropin regulation of PACAPR gene expression during follicle growth in the rat ovary. We report here the stage-specific expression and gonadotropin induction of PACAPR mRNA in granulosa cells during ovarian follicle growth. We also demonstrated that the short variant form of PACAPR was predominant in the ovary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immature female rats of the Sprague Dawley strain were purchased from Daehan Laboratories (Chungbuk, Korea). They were housed in groups in a room with controlled temperature and photoperiod (10-h dark, 14-h light, with lights on from 0600–2000 h). The animals had ad libitum access to food and water. The animals, ranging in age from 3–21 days, were killed by cervical dislocation, and the ovaries were removed for RNA analysis. Ovaries were also collected from immature (26-day-old) rats at various times after treatment with 10 IU PMSG to induce multiple follicle growth. 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.
Northern blot analysis
Total RNA from ovaries or cultured follicles was isolated using
Tri Reagent solution (Sigma). Twenty micrograms of total
RNA were fractionated by electrophoresis on a 1% agarose gel
containing formaldehyde and transferred to nylon membranes by capillary
blotting with 10 x sodium citrate-sodium chloride (SSC). After a
UV cross-linking and prehybridization, membranes were hybridized
overnight at 42 C in a solution containing 50% formamide, 5 x
SSC, 1 mM EDTA, 250 µg/ml denatured salmon sperm DNA, 500
µg/ml yeast transfer RNA, and a total of 1 x
107 cpm of a 32P-labeled
rat PACAPR complementary DNA (cDNA) probe (20). 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 7–10 days at
-80 C. The band intensities were subsequently measured using a
phosphorimager (Bio-Rad Laboratories, Inc. Hercules, CA),
and the signals were normalized to the 28S ribosomal RNA internal
control.
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)-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 (9). 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 PACAPR complementary
RNA (cRNA) probe (2 x 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 and developed after 3–4 weeks. The slides were stained with
hematoxylin and eosin and examined under the light microscope with
bright- and dark-field illumination.
Follicle culture
Preovulatory follicles (>800 µm in diameter) were dissected
by fine forceps from ovaries collected at 48–52 h after PMSG
injection, and follicle culture was performed as previously described
(9). Fifteen to twenty follicles were cultured in glass vials
containing 800 µl MEM (Life Technologies, Inc., Grand
Island, NY) supplemented with penicillin, streptomycin,
L-glutamine, and 0.1% BSA (wt/vol, Fraction V,
Sigma) in the absence or presence of LH. Cultures were
maintained for up to 24 h at 37 C under 5%
CO2-95% O2. Following
incubation, follicles were snap-frozen for RNA isolation, or were fixed
for in situ hybridization analysis.
RNase protection assay
For the detection of the expression of different PACAPR variants
in the ovary, RNase protection assay (RPA) was performed using RPA II
Kit (Ambion, Inc., Austin, TX) with cRNA probe
encompassing the splice site of PACAPR (nucleotides 793-1143) (12). The
expected bands after RNase digestion include 351 bp for the short
variant form and 248/103 bp for other longer variant forms. Run-off
transcripts were synthesized from each linearized template using a
Transcription in vitro System Kit (Promega Corp., Madison, WI). The full-length single-stranded RNA probes
were purified by acrylamide gel electrophoresis. Twenty to thirty
micrograms of ovarian total RNA were hybridized with
32P-labeled rat PACAPR and rat
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cRNA probes at 43 C
overnight. After hybridization samples were digested with RNase A and
T1 before precipitation and electrophoresis on a 5% denaturing
polyacrylamide gel. After electrophoresis, gels were exposed to the
Kodak RX films for 48–72 h at -80 C. The band
intensities were subsequently measured using a phosphorimager
(Bio-Rad Laboratories, Inc.), and the signals were
normalized to the GAPDH internal control.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the 7-kb transcript was detected in
the ovary as well as in the hypothalamus. The ovarian PACAPR transcript
was visible in 3-day-old rats and markedly increased in 21-day-old
rats. The levels of ovarian PACAPR expression were 2.2-fold higher in
21-day-old rats than in 15-day-old rats (Fig. 1B). To determine the
site of PACAPR mRNA expression, in situ hybridization was
performed on ovarian sections obtained from 21-day-old rats. PACAPR
mRNA was detected in growing follicles of immature rat ovaries (Fig. 2). High levels of PACAPR mRNA were
mainly detected in large preantral follicles (PAF) (Fig. 2, A and B).
Some small preantral follicles also expressed PACAPR mRNA (Fig. 2, A
and B). Under higher magnification, the PACAPR signal was shown to be
restricted to the granulosa cells (Gc) of preantral follicles (Fig. 2C). However, atretic follicles (AtF) and mural granulosa cells of
early antral follicles (EAF) were devoid of PACAPR mRNA (Fig. 2, A and
B). No specific signal was detected in ovarian sections hybridized with
sense probe (Fig. 2D).
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, PMSG treatment of 26-day-old
immature rats caused a gradual decrease in ovarian PACAPR expression.
Treatment with hCG of PMSG-primed rats, however, stimulated ovarian
PACAPR expression within 3–6 h. The levels of PACAPR mRNA declined by
9 h after treatment. Quantitative analysis of ovarian PACAPR
signals (Fig. 3B) showed a 55% decrease 48 h after PMSG and a
160% increase 6 h after hCG. To determine the cell types
expressing PACAPR mRNA in gonadotropin-treated ovaries, antisense and
sense cRNA probes for PACAPR were generated for in situ
hybridization analysis. In ovaries obtained 2 days after PMSG
treatment, PACAPR signals were detected in growing follicles, but not
in preovulatory follicles (Fig. 4A). In
the ovaries of PMSG-primed immature rats, followed by hCG stimulation
for 6 h, PACAPR mRNA was detected in preovulatory follicles as
well as in growing follicles (Fig. 4B). Under higher magnification, the
PACAPR signal was shown to be defined in the granulosa cells of
preovulatory follicles (Fig. 4C). Ovarian sections hybridized with the
sense PACAPR riboprobe showed only background hybridization (Fig. 4D).
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, left panel).
Furthermore, LH treatment for 6 h stimulated PACAPR mRNA in a
dose-dependent manner, reaching saturating levels at 100 ng/ml (Fig. 5A, right panel). Quantitative analysis (Fig. 5B)
showed that LH treatment resulted in 3.4- and 3.0-fold increase in
follicular PACAPR mRNA by 6 h and 9 h, respectively. To
determine the site of PACAPR mRNA expression in cultured preovulatory
follicles, in situ hybridization was performed on sections
of follicles incubated in the presence of LH for 6 h. PACAPR
signals were detected in granulosa cells, but not theca cells (Fig. 6, A and B). Under higher magnification,
patchy signals of PACAPR mRNA were seen in the granulosa cells of
preovulatory follicles (Fig. 6C). Preovulatory follicles cultured in
the absence of LH for 6 h did not express PACAPR mRNA (data not
shown). No specific signal was detected in follicles hybridized with
sense probe (Fig. 6D).
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protected band of the short variant form (short; 351 bp) and other
longer variant forms (hip/hop; 248 bp) of PACAPR were found to be
present in ovaries of 15-day-old rats. However, only the expression of
the short variant form was clearly increased in ovaries of 21-day-old
rats. Furthermore, treatment of PMSG-primed rats with hCG resulted in
the stimulation of the short variant form, but not longer variant
forms. Similarly, inclusion of LH in the culture medium of preovulatory
follicles caused up-regulation of the short variant form of PACAPR.
Both the short and longer variant forms of PACAPR were expressed in the
brain which was used as a positive control (Fig. 7A). Figure 7B
graphically summarizes the quantitative densitometry results of these
studies after normalization for glyceraldehyde-3phosphate dehydrogenase
(GAPDH) mRNA levels. The levels of the short variant form of PACAPR
were increased, on the average, 4.6-, 2.9-, and 5.1-fold in ovaries of
21-day-old rats, in PMSG/hCG-treated ovaries and in preovulatory
follicles cultured in the presence of LH compared with the
corresponding control levels, respectively. In contrast, the levels of
the longer variant forms of PACAPR did not significantly vary with the
age of rats or gonadotropin treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PACAP can interact with specific receptors. The type I receptor (PACAPR) specifically binds PACAP, whereas type II receptor (VIPR) binds PACAP and VIP with similar affinity (21). VIP-immunoreactive nerve fibers are detected in theca/interstitial cells of the juvenile rat ovary (22), and VIP has been shown to increase cAMP accumulation and steroidogenesis in cultured granulosa cells (23). Although binding studies using whole ovaries have suggested the presence of VIPR in the rat ovary (19), a recent paper demonstrates the expression of PACAPR mRNA in the ovary of adult cycling rats of undocumented cycle stage (16). Furthermore, PACAP has been shown to be more potent than VIP in the stimulation of steroidogenesis and cAMP accumulation in cultured granulosa cells of large growing follicles obtained from estrogen-treated immature rats (7, 8), suggesting that these cells may contain PACAPR. The present study provides the direct evidence for the presence of PACAPR in granulosa cells of growing follicles. Our observations showing low levels of PACAPR expression in ovaries of prepubertal rats before 15 days of age and a marked increase at 21 days of age indicate that PACAPR expression is increased in a stage-specific manner during ovarian follicle growth. It has been shown that ovaries of rat before 15 days of age mainly contain nongrowing and small growing follicles (24). The stage-specific expression of PACAPR is further supported by the fact that PMSG treatment caused time-dependent decrease in ovarian PACAPR expression. PMSG is a hormone known to stimulate a multiple growth of growing follicles to the preovulatory stage (25). Of further interest are the present findings demonstrating the stimulation of PACAPR mRNA in granulosa cells of preovulatory follicles by LH/hCG. Support for our findings of gonadotropin stimulation of PACAPR in preovulatory follicles comes from a recent report showing that cotreatment with PACAP-38 and LH results in the higher production of progesterone and cAMP than treatment with PACAP-38 or LH alone in cultured granulosa cells obtained from rat preovulatory follicles (17). Thus, the present findings suggest that the direct PACAPR-mediated actions of PACAP on ovarian function may be restricted to specific developmental windows.
Our previous study, demonstrating the gonadotropin induction of PACAP in granulosa cells of preovulatory follicles and in some theca/interstitial cells regardless of follicle size, has suggested a role of PACAP as an ovarian local regulator (9). Data from the present study provide further evidence that PACAP may participate in the regulation of ovarian function. First, it may act as a mediator for LH action during ovulation. The LH surge is obligatory to induce the processes of follicle rupture and luteinization. Although the biochemical cascade that leads to ovulation is largely unknown, a number of specific genes associated with ovulation have been identified (26). The rapid, but transient, increase in PG endoperoxide synthase (27), tissue plasminogen activator (28), and progesterone receptor (29) has been shown to play a role in LH-induced ovulation. The similar observation that LH induces a transient expression of both PACAP (9) and PACAPR in granulosa cells of preovulatory follicles raises the possibility that PACAP may be functionally associated with the process of follicle rupture and/or luteinization, acting as an autocrine regulator. Indeed, a recent report demonstrates an autocrine role for PACAP during gonadotropin-induced periovulatory progesterone production and subsequent luteinization in the rat ovary (30). Second, PACAP may have a role in the regulation of early folliculogenesis. It is known that ovarian follicular growth begins and proceeds to the late preantral stage independently of gonadotropin regulation (31). Further development depends upon FSH acting upon its cognate receptor expressed by granulosa cells (32). It has been suggested that insulin-like growth factor I, expressed in granulosa cells of healthy growing follicles, may promote follicle growth by augmenting granulosa cell FSH receptor responsiveness (33), and thereby amplifying FSH-induced aromatase expression and LH receptor induction (34, 35). Similarly, activin has been shown to promote folliculogenesis during the preantral or early antral stages of growth of follicles by amplifying granulosa cell responsiveness to FSH (36). The present study demonstrating the major expression of PACAPR in granulosa cells of large preantral follicles indicates that PACAP may promote the growth of these preantral and early antral follicles. PACAP secreted from granulosa cells of preovulatory follicles and/or from theca cells of growing follicles after LH/hCG stimulation (9) may bind to its receptors present in granulosa cells of growing follicles, and hence may enhance granulosa cell responsiveness to FSH. This action of PACAP following LH surge in cyclic rats may be needed to stimulate follicle recruitment for the subsequent cycle by enhancing the effect of a secondary surge of FSH on the day of estrus, which has been suggested to play a role in follicular recruitment (37). Transient induction of PACAP after the LH surge has also been demonstrated in cyclic rats (38). Studies are currently underway to examine these possibilities for the role of PACAP in the process of ovulation and in the promotion of growth of growing follicles.
Five splice variants of the PACAPR with different pattern of signal transduction have been identified in the rat (12). The short PACAPR (without hip/hop cassette) as well as the PACAPR hop variants potently activate both adenylate cyclase and phospholipase C, whereas the PACAPR hip and hip-hop variants display a deficient or altered ability to activate phospholipase C. Moreover, the splicing of the PACAPR gene is differentially regulated in adult tissues. The short PACAPR is the most abundant form in the brain, whereas PACAPR hop predominates in the testes, olfactory bulb, and adrenal gland (12). The present study demonstrates that the short PACAPR was the predominant form stimulated during prepubertal development and by LH/hCG during ovarian follicle growth. The expression of PACAPR splice variants in adult rat ovary has also been identified by the techniques of RT and PCR (RT-PCR) (16). Because splice variants of the PACAPR display differential signal transduction properties in the same cell type (12), the expression of different variants of PACAPR may subserve distinct patterns of signal transduction activated by PACAP during ovarian follicle growth. Further studies are necessary to ascertain whether the expression of the short PACAPR variant is physiologically important in the ovary during prepubertal development and gonadotropin-induced ovulation.
In summary, the present study has demonstrated that PACAPR gene is expressed in the rat ovary in a stage- and cell-specific manner during follicle development. The increased expression of PACAPR, mainly the short variant form, is restricted to granulosa cells of the growing follicles during prepubertal development and to granulosa cells of preovulatory follicles after gonadotropin stimulation. Further studies are needed to determine whether PACAP is involved in the regulation of early follicle growth and ovulation during follicle development.
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Footnotes |
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Received June 8, 1999.
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