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Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5
Address all correspondence and requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynecology, University of British Columbia, 2H-30, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}interchange.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to its well-documented role in gonadotropin biosynthesis and secretion, GnRH-I has been implicated in the endocrine functions of the gonads. This concept is based on the detection of GnRH-I gene transcripts, synthesis of the GnRH-I, and the multitude of effects attributed to GnRH receptor (GnRHR)-mediated signaling in extrapituitary tissues (8, 9, 10, 11, 12). In the ovary, GnRH-I modulates both basal and gonadotropin-stimulated steroidogenesis (13, 14) and induces transcription of several genes involved in the follicular maturational process and ovulation (15, 16). However, the presence and role of a second form of GnRH in gonads have not been reported.
Physiologic studies have indicated that levels of GnRH-I and GnRHR are regulated by several hormones, including its homologous ligand GnRH-I and gonadotropins (1). In the hypothalamus, the synthesis and release of GnRH-I is regulated by numerous hormones, either directly (involving GnRH neurons) or indirectly (involving neurons that communicate with GnRH neurons) (17). GnRH-I has been shown to regulate its own ligand in a biphasic manner in immortalized hypothalamic GT1–7 neurons (18) and normal ovarian surface epithelium (OSE) (19). The treatment of GT1–7 neurons with human CG (hCG) resulted in a dose- and time-dependent decrease in the steady-state GnRH-I (20). It is well documented that the number and messenger RNA (mRNA) levels of GnRHR are both up- and down-regulated by its homologous ligand in the pituitary and ovary (1, 12, 13, 21). In addition to their own ligand, gonadotropins have been shown to regulate the levels of GnRHR mRNA in the ovary (12, 14). However, the hormonal regulation of GnRH-II and its influence on GnRHR levels in the ovary remain to be elucidated.
The present study was designed to investigate the physiological role of GnRH-II and its hormonal regulation by homologous ligand (GnRH-II) and gonadotropins (FSH and hCG) in the human ovary.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
The use of human granulosa-luteal cells (hGLCs) was approved by
the Clinical Screening Committee for Research and Other Studies
involving Human Subjects of the University of British Columbia.
Follicular aspirates were collected during oocyte retrieval from women
undergoing in vitro fertilization. Human GLCs were prepared
as previously described (22) and cultured in DMEM
supplemented with 10% FBS, 100 U/ml penicillin G, and 100 µg/ml
streptomycin, for 4 days before treatment in a humidified atmosphere of
5% CO2-95% air at 37 C.
Treatments
To investigate homologous or heterologous regulation of GnRH-II
mRNA levels, hGLCs were cultured for 4 days and then incubated in 2 ml
DMEM containing 5% FBS, GnRH-II
(10-11–10-7
M), GnRH-II-a
(10-11–10-7
M), GnRH-I-a
(10-11–10-7
M), rFSH (0.1–1000 ng/ml), or hCG (0.001–10 IU/ml) in a
humidified atmosphere of 5% CO2-95% air at 37
C. After 24 h incubation, medium was collected and stored at -20
C and subsequently assayed for progesterone content. Cells were lysed
and thereafter immediately frozen at -70 C until total RNA was
extracted.
Total RNA isolation and RT-PCR amplification
Total RNA was prepared from cultured cells using the RNaid kit
(Bio/Can Scientific, Mississauga, Canada) according to the
manufacturer’s suggested procedure. The RNA concentration was measured
based on the absorbance at 260 nm, and its integrity was confirmed by
agarose-formaldehyde gel electrophoresis. Total RNA (1 µg) was
reverse transcribed into first-strand cDNA (Amersham Pharmacia Biotech), following the manufacturer’s procedure. To clone
GnRH-II mRNA, one set of primers was designed based on the published
sequences for human GnRH-II (4). Primers for GnRH-II were:
sense, 5'-GCCCACCTTGGACCCTCAGAG-3'; and antisense,
5'-CCAGGTGTCGCTTCCTGTGAA-3'. The cDNA was amplified in a 50-µl PCR
reaction containing 2.5 U Taq polymerase and its buffer, 1.5
mM MgCl2, 2
mM deoxynucleotide triphosphate, and 50 pmol
specific primers. PCR amplification was carried out for 33 cycles with
denaturing for 1 min at 94 C, annealing for 35 sec at 60 C, extension
for 90 sec at 72 C, and a final extension for 15 min at 72 C.
Cloning and sequencing of RT-PCR product
Ten microliters of PCR products were fractionated on a 1.5%
agarose gel stained with ethidium bromide. The expected PCR product
(225-bp) was isolated from the gel and cloned into the pCRII vector
using the TA Cloning Kit (Invitrogen, San Diego, CA).
Positive clones were isolated and sequenced by the dideoxy nucleotide
chain termination method using the T7 DNA polymerase sequencing kit
(Amersham Pharmacia Biotech). Sequence analysis of the
cDNA revealed that GnRH-II cDNA has identical sequence to those from
the published human GnRH-II. This cDNA was then used as the template
for making digoxigenin-labeled probe for Southern blot analysis.
Quantification of GnRH-II, GnRH-I, GnRHR, FSHR, and LHR mRNA
To compare different expression levels for GnRH-II mRNA,
semiquantitative PCR was performed. Primers for GnRH-II were: sense,
5'-GCCCACCTTGGACCCTCAGAG-3'; and antisense,
5'-CCAATAAAGTGTGAGGTTCTCCG-3'. PCRs for GnRH-II were carried out with
denaturing for 1 min at 94 C, annealing for 65 sec at 62 C, extension
for 90 sec at 72 C, and a final extension for 15 min at 72 C for 26
cycles. PCRs for GnRH-I, GnRHR, and ß-actin were performed as
described previously (12, 22). PCR for ß-actin was
performed to rule out the possibility of RNA degradation and was used
to control the variation in mRNA concentration in the RT reaction, as
previously described (23). Semiquantitative PCRs for FSHR
and LHR were performed. The primers for LHR were: sense,
5'-GCCCACCTTGGACCCTCAGAG-3'; and antisense,
5'-CCAATAAAGTGTGAGGTTCTCCG-3'. Primers for FSHR were described
previously (24). PCRs for FSHR and LHR were carried out
with denaturing for 1 min at 94 C, annealing for 35 sec at 55 C,
extension for 90 sec at 72 C, and a final extension for 15 min at 72 C
for 25 cycles. Ten microliters of PCR products were fractionated on a
1.5% agarose gel stained with ethidium bromide. The PCR products were
transferred to a nylon membrane (Hybond-N) and hybridized with
digoxigenin-labeled cDNA probes for human GnRH-II, GnRH-I (12, 22), GnRHR (12, 22), ß-actin (23),
FSHR (25), and LHR (26), following the
manufacturer’s recommended procedure (Roche Molecular Biochemicals, Laval, Canada). The digoxigenin-labeled cDNAs for
GnRH-II, GnRH-I, GnRHR, FSHR, LHR, and ß-actin were prepared using
DIG-DNA Labeling Kit according to manufacture’s suggested procedure
(Roche Molecular Biochemicals). After washing, the signals
were detected with anti-DIG-conjugated secondary antibody and
visualized using the CSPDR chemiluminescent
system (Roche Molecular Biochemicals), followed by
autoradiography. The intensities of signal were quantified by
densitometry using NIH Image ß 3. The expression levels of GnRH-II,
GnRH-I, FSHR, and LHR mRNA were normalized against ß-actin mRNA
levels. The amount of GnRHR transcript was calculated from the ratio of
the target to competitive cDNA, as described previously
(22).
RIA for progesterone
To investigate the role of GnRH-II on basal progesterone
secretion, hGLCs were cultured for 4 days and then incubated for
24 h in 2 ml DMEM containing 5% FBS,
10-11–
10-7 M of
GnRH-II, and GnRH-II-a in a humidified atmosphere of 5%
CO2-95% air at 37 C. To examine the effect of
GnRH on hCG-stimulated progesterone secretion, cell cultures were
treated with GnRH-II (10-7
M), GnRH-II-a
(10-7 M), or
GnRH-I-a (10--7
M) in the presence or absence of hCG (1 IU/ml) for 24
h. The appropriate cell cultures were treated with GnRH-II-a
(10-7 M) plus
the GnRH antagonist (antide,
10-7 M) for
24 h to determine whether the effect of GnRH-II is mediated
through the activation of classical GnRHR. Control cultures were
treated with vehicle. After 24 h incubation, the culture medium
was collected, and cells were lysed with RIPA [150 mM
NaCl, 1% Nondiet P-40, 0.5% deoxycholate, 0.1% SDS, 50
mM Tris (pH, 7.5) and 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 100 µg/ml
aprotinin]. The progesterone concentration in the culture medium was
measured by an established RIA, as previously described
(27). Intra- and interassay variation coefficients were
5% and 7%, respectively. Protein content in the cell lysate was
determined using the Bio-Rad Laboratories, Inc. protein
assay kit (Richmond, CA), according to the manufacture’s suggested
procedure, and was used to standardize progesterone secretion.
Progesterone secretion was expressed as the percent change relative to
the control value.
RIA for intracellular cAMP
To determine whether GnRH-II modulates basal and hCG-stimulated
intracellular cAMP accumulation, hGLCs (2 x
105 cells) were plated onto 35-mm culture dishes
and cultured for 4 days. The cells were then preincubated in serum-free
medium containing 0.1% BSA and 0.5 mM IBMX, for 30 min,
and treated with GnRH-II
(10-7 M) or
GnRH-II-a (10-7
M) in the presence or absence of hCG (1 IU/ml) for 20 min.
Control cultures were treated with vehicle. Intracellular cAMP levels
were measured using a [3H]-cAMP assay system
(Amersham Pharmacia Biotech) according to the
manufacturer’s suggested procedure.
Data analysis
Data are shown as the means of four individual experiments and
are presented as the mean ± SD. The data were
analyzed by one-way ANOVA followed by Tukey’s multiple-comparison
test. P < 0.05 was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Twenty-six cycles for GnRH-II and 25
cycles for FSHR and LHR were employed for quantification. The
validation of PCRs for GnRH-I, GnRHR, and ß-actin was described
previously (12, 22).
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). Similarly, GnRH-II-a down-regulated
GnRH-II mRNA levels at all concentrations used
(10-11–10-7
M), with maximum down-regulation (63% of control levels)
at 10-9 M
(Fig. 2). As shown in Fig. 3, both
GnRH-II and GnRH-II-a decreased GnRHR mRNA levels at all concentrations
used
(10-11–10-7
M), with maximum down-regulation at
10-10 M (42%
and 40% of control levels, respectively). In contrast, treatment with
GnRH-I-a resulted in a biphasic effect for GnRH-I and GnRHR mRNA
levels. High concentrations of GnRH-I-a
(10-8 and
10-7 M)
decreased GnRH-I and GnRHR mRNA levels, whereas low concentrations
(10-11 and
10-10 M)
resulted in an up-regulation of GnRH-I and its receptor (Figs. 2B and 3B).
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, treatment with
FSH resulted in a dose-dependent increase in GnRH-II mRNA levels, with
maximum increase (347% of control levels) at 1000 ng/ml. Similarly,
treatment with hCG resulted in a significant up-regulation of GnRH-II
mRNA levels, with maximum increase (357% of control levels) at 0.01
IU/ml (Fig. 4B). In contrast, treatment with FSH and hCG resulted in a
down-regulation of GnRH-I mRNA levels, with maximum down-regulation at
10 ng/ml FSH (63% of control levels) and 1 IU/ml hCG (42% of control
levels) (Fig. 4). Functionally, treatment with FSH and hCG stimulated
progesterone secretion from hGLCs (data not shown).
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). Maximum
inhibition (a 33% decrease over basal level) was observed after the
treatment with 10-8
M GnRH-II. A further decrease in progesterone secretion was
observed in cells treated with
10-10–10-7
M GnRH-II-a, with maximum inhibition (a 49% decrease over
basal levels) at 10-7
M GnRH-II-a (Fig. 5A). Similarly, treatment with
10-7 M
GnRH-I-a resulted in a significant decrease in progesterone secretion
(a 52% decrease over basal levels) (Fig. 5B). hCG alone (1 IU/ml)
stimulated progesterone secretion (a 235% increase over basal levels),
whereas concomitant treatment with GnRH-I-a or GnRH-II-a attenuated the
stimulatory effect of hCG (Fig. 5B). Cotreatment with antide abolished
the inhibitory effect of GnRH-II-a on progesterone secretion (Fig. 5C),
whereas antide alone had no effect on progesterone secretion (Fig.
5C).
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, treatment of
hGLCs with various concentrations of GnRH-II and GnRH-II-a resulted in
a significant decrease in FSHR mRNA levels, with maximum decrease at
10-9 M of
GnRH-II (35% of control levels) and
10-7 M of
GnRH-II-a (35% of control levels). Similarly, a down-regulation of
FSHR mRNA levels was observed in cells treated with GnRH-I-a at all
concentrations used, maximum decrease (41% of control levels) at
10-9 M (Fig. 6A). Like FSHR mRNA, GnRH-II or GnRH-II-a decreased LHR mRNA levels,
with a maximum down-regulation at
10-10 M of
GnRH-II (68% of control levels) and at
10-10 M of
GnRH-II-a (68% of control levels) (Fig. 6B). A similar decrease in LHR
mRNA levels was observed in cells treated with GnRH-I-a, with a maximum
decrease (52% of control levels) at
10-8 M (Fig. 6B).
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). Concomitant treatment of cells with
10-7 M GnRH-II
or GnRH-II-a did not affect hCG-stimulated cAMP production (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the interesting findings of the present study is the demonstration of differential regulation of GnRH-II gene expression by GnRH-II itself and gonadotropins, compared with GnRH-I. GnRH-I has been shown to exert biphasic effects on GnRH-I secretion from immortalized and normal hypothalamic neurons, depending on the concentration and duration of treatment (18, 28). As in the hypothalamus, GnRH-I has been shown to regulate its own mRNA level in a biphasic manner in human OSE cells (19). As well, it has been well documented that GnRH-binding sites are both up- and down-regulated by GnRH-I in the pituitary of various species (1, 21). In general, low doses or pulsatile treatment of GnRH-I up-regulate its receptor, whereas high doses or continuous treatment down-regulate the receptor numbers. Changes in the GnRHR mRNA levels have been explained as at least part of the mechanisms underlying up- and down-regulation of GnRHR numbers. Pulsatile treatment with 10 nM GnRH-I resulted in a decrease of GnRHR mRNA levels in rat pituitary cells (29), whereas continuous treatment with the same concentration of GnRH-I for 48 h decreased levels of the receptor mRNA in cultured sheep pituitary cells (30). A biphasic regulation pattern of GnRHR mRNA levels has been reported in hGLCs (12) and human OSE cells (19). In the present study, a biphasic response of GnRH-I and GnRHR mRNA was observed in response to GnRH-I-a treatment, confirming previous studies. Low doses of GnRH-I-a (10 and 100 pM) increased GnRH-I and GnRHR mRNA levels, whereas higher doses (10 and 100 nM) of the GnRH-I-a resulted in a significant decrease in GnRH-I and GnRHR mRNA levels. In contrast to GnRH-I, no biphasic response was observed in response to GnRH-II. Treatment with GnRH-II, at all concentrations used (10 pM to 100 nM), resulted in a significant down-regulation of both GnRH-II and GnRHR mRNA levels. The exact mechanism for this differential regulation is not clear. It is possible that GnRH-I and GnRH-II may have different binding characteristics for GnRHR, which may cause distinct receptor conformations. These ligand-specific conformations of the GnRHR in hGLCs could lead to differential coupling to G proteins and/or generate different intracellular signal transduction pathways, eventually leading to differential regulation of GnRH and its receptor gene expression. In this regard, GnRHR has been shown to couple with different subtypes of G proteins, leading to activation of differential intracellular signaling pathways in the same or distinct cells (31, 32, 33, 34). Furthermore, two native GnRH forms in goldfish (sGnRH and cGnRH-II) have been shown to activate differential signal transduction pathways that differ in their relative dependence on intracellular and extracellular Ca2+ availability, protein kinase C activation, inositol phosphate production, and arachidonic acid mobilization (35, 36, 37, 38).
In addition to its own ligand, gonadotropins have been shown to regulate GnRH-I mRNA expression. It has been demonstrated that hCG decreases GnRH-I mRNA in GT1–7 neuron in a dose- and time-dependent manner (20), supporting the presence of a short feedback mechanism (39, 40). In the present study, treatment of hGLCs cells with FSH and hCG resulted in a marked increase in GnRH-II mRNA levels but decreased GnRH-I mRNA. Several studies have demonstrated the differential regulation of two forms of GnRH during various physiological conditions. In the brain of the European female silver eel, steroids control differential regulation of two forms of GnRH, with a positive estrogen-dependent feedback on mGnRH and a negative androgen-dependent feedback on cGnRH-II (41). As well, in the chicken, only the cGnRH-I levels in the hypothalamus change with castration (42). In the goldfish, the ratio between sGnRH and cGnRH-II changes with sexual maturation (43). A stronger increase in sGnRH than in cGnRH-II has been observed in the pituitary. Taken together, the differential regulation of two forms of GnRH by gonadotropins, in the present study, suggest that gonadotropins may regulate the ratio between GnRH-I and GnRH-II, leading to distinct spatial expressions of these peptides. However, the physiological relevance of this differential regulation remains to be determined.
Increasing evidence has suggested that two or three identified forms of GnRH in a single species may have a similar physiological role (3, 44, 45). Administration of synthetic GnRH-II to adult rhesus monkeys resulted in a significant increase in the plasma LH concentration, suggesting that GnRH-II may also have a physiological role in regulating the release of LH (3). In the goldfish, the two endogenous forms of GnRH stimulate the release of both gonadotropins and growth hormones from the pituitary, even though there are functional differences in terms of potency (7, 42, 45). In adult female sea lampreys, two endogenous forms of GnRH (lamprey GnRH-I and -III) have been shown to stimulate ovarian steroidogenesis (45). Like GnRH-I, in the present study, GnRH-II inhibited both basal and hCG-stimulated progesterone secretion in hGLCs. This result suggests that GnRH-II has a similar biological role with respect to ovarian steroidogenesis. However, we cannot rule out the possibility that GnRH-II may have other unique reproductive functions, compared with GnRH-I, in the ovary. The inhibitory effect of GnRH-II on progesterone secretion, in the present study, seems to be mediated via activation of the classical GnRHR, because cotreatment with antide abolished the effect of GnRH-II. However, it is possible that GnRH-II may bind and activate on unknown second type of GnRHR whose activation may also be blocked by antide. Even though a second form of GnRHR has not been demonstrated in the human, recent cloning of two GnRHR subtypes, with distinct ligand selectivity in the goldfish, supports this notion (46). Furthermore, binding studies have indicated the presence of two different types of GnRHR in the ovary, as high-affinity-low-capacity and low-affinity-high-capacity GnRH binding sites (11, 47).
The antigonadotropic action of GnRH-I has been shown to be mediated through a down-regulation of receptors for FSH and LH (48, 49), inhibition of gonadotropin-stimulated cAMP production (50, 51), and steroidogenic enzymes (52, 53). Like GnRH-I, the treatment of hGLCs with GnRH-II resulted in a significant down-regulation of FSHR and LHR. In contrast, GnRH-II did not affect basal and hCG-stimulated cAMP production. Our previous results showed that GnRH-I also had no effect on basal and hCG-stimulated cAMP production (54). These results suggest that GnRH-II, like GnRH-I, exerts its antigonadotropic effect at the receptor levels in hGLCs, independent of cAMP levels.
In summary, we have demonstrated that GnRH-II and GnRH-I are under differential regulation by their homologous ligands and gonadotropins. Nevertheless, both forms of GnRH seem to have similar effects in the ovary, especially with regard to alterations in gonadotropin receptor mRNA levels and steroidogenesis.
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Acknowledgments |
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Footnotes |
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2 Recipients of a studentship award from the British Columbia
Research Institute of Children’s and Women’s Health.
3 A career investigator for the British Columbia Research Institute
of Children’s and Women’s Health.
Received July 31, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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