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Original Studies |
-Chain Messenger Ribonucleic Acids That Are Regulated by GnRH in Vitro1
Department of Obstetrics and Gynecology, National Taiwan University Hospital (H.-F.C.), Taipei, Taiwan; the Laboratory of Veterinary Biochemistry, Chungbuk National University (E.-B.J.), Cheong-ju, Korea; and the Department of Obstetrics and Gynecology, University of British Columbia (M.S., P.C.K.L.), Vancouver, British Columbia, Canada V6H 3V5
Address all correspondence and requests for reprints to: Dr. P. C. K. Leung, Department of Obstetrics and Gynecology, 4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: peleung{at}unixg.ubc.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain messenger ribonucleic acids (mRNAs) in human peripheral blood
mononuclear cells. It was found that human mononuclear cells expressed
GnRH and GnRHR mRNAs. Nucleotide sequences of these mRNAs are identical
to their hypothalamic and pituitary counterparts, respectively. In
addition, GnRH and GnRHR mRNA expressions in peripheral blood
mononuclear cells are regulated by GnRH and its synthetic analogs
in vitro. Treatment with various concentrations of GnRH
(10-5-10-11 mol/L) increased GnRHR mRNA
expression in a dose-dependent manner (maximal level is 158% of the
untreated control value at 10-8 mol/L GnRH;
P < 0.05), but reduced GnRH mRNA levels to 69% of
the untreated control value at 10-9 mol/L GnRH
(P < 0.05). Cotreatment of GnRH with a GnRH
antagonist blocked these regulatory effects, indicating the
receptor-mediated nature of the GnRH action. Both GnRH and GnRH
agonist stimulated interleukin-2 receptor -chain mRNA in a
dose-dependent manner, indicating that GnRH may be involved in
lymphocyte activation. In summary, these observations suggest that
mRNAs encoding the pituitary form of GnRHR and the hypothalamic form of
GnRH are also expressed in human peripheral blood mononuclear cells.
The endogenous production of GnRH by lymphocytes may act as an
autocrine or paracrine factor to regulate immune functions. Because of
the presence of GnRHR on lymphocytes, exogenous GnRH analog therapy may
have an impact on the immune system through these receptors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GnRHR is a member of the G protein-coupled receptor superfamily containing seven transmembrane domains. Previous studies of GnRHR were limited to ligand binding assays. Recently, both human and mouse pituitary GnRHR complementary DNA (cDNAs) have been cloned (2, 3, 4, 5, 6), and the human GnRHR gene has been mapped to chromosome 4 (7). There is evidence suggesting that GnRHR is not limited to pituitary gonadotropes (8); for example, GnRHR or GnRH-binding sites have been identified in tissues including the ovary, testis, breast, and prostate (2, 9, 10, 11, 12) as well as in breast ovarian and endometrial tumors (11, 13). In addition, GnRH-binding sites have been reported in lymphoid tissues such as mouse (14) and rat spleen (15), rat thymus (15, 16, 17), and porcine lymphocytes (18). The GnRHR or GnRH-binding sites in lymphocytes are probably functional, as previous studies have demonstrated that GnRH agonist (GnRH-a) caused a dose-dependent increase in LH production, whereas GnRHR antibodies blocked this action (14). It has also been shown that blockade of central and peripheral GnRHR with a potent GnRH antagonist (GnRH-ant) during a critical period of maturation can impair the morphological development of thymus and the cellular and humoral immune responses in rats (17) and monkeys (19). GnRH-like substances have been reported to be secreted from tissues outside the central nervous system of rats (20, 21, 22) and humans (23). GnRH messenger ribonucleic acid (mRNA) has been detected in rat lymphocytes (24) and human ovary (11, 25). The presence of both the ligand (GnRH-like substance) and the receptor (GnRH-binding site) in cells from the immune system indicates the potential function of GnRH in the immune system. To our knowledge, the expression and regulation of GnRH and its receptor at the mRNA level in human peripheral lymphocytes have not been studied. A comparison of the authenticity of these mRNAs with their pituitary and hypothalamus counterparts has not been reported. These issues are carefully examined in this study.
In recent years, GnRH analogs have become widely used in both benign and malignant disorders, such as precocious puberty, endometriosis, uterine leiomyoma, infertility, and certain hormone-dependent tumors. Relatively little is known about the side-effects of GnRH analogs in humans, especially in relation to the immune system. Thus, the present study was designed to identify the presence of GnRH and GnRHR in human peripheral blood mononuclear cells. Secondly, the regulation of GnRH and GnRHR expression by exogenous GnRH and its derivatives were examined. Finally, the effect of exogenous GnRH on the expression of interleukin-2 receptor (IL-2R) mRNA, one of the major markers of lymphocyte activation, was studied.
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Materials and Methods |
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The use of human mononuclear cells for research was approved by University of British Columbia clinical screening committee for research and other studies involving human subjects. Fresh human venous blood samples from female donors, aged 19–34 yr, were obtained. These blood samples were negative for cytomegalovirus antibody (Ab), human T-cell lymphoma virus and II Abs, human immunodeficiency virus I p24 antigen, hepatitis B surface antigen, hepatitis C Ab, and the Venereal Disease Research Laboratories test. The blood samples were processed between 1–3 days after donation. The Ficoll-Hypaque method was used to isolate mononuclear cells from the blood. Briefly, the blood was first diluted 1:1 with phosphate-buffered saline, and 10 mL diluted blood were carefully layered onto a 3-mL Ficoll-Haque Plus cushion (Pharmacia Biotech, Uppsala, Sweden) in a 15-mL centrifuge tube (Falcon 3033 Becton Dickinson, Franklin Lakes, NJ). The tube was centrifuged at 400 x g for 30 min at 18–20 C. The interface (containing mononuclear cells) was carefully collected and washed twice with phosphate-buffered saline and once with RPMI 1640 medium (Life Technologies, Grand Island, NY) with 1% FBS. The cell pellet was resuspended in 5 mL culture medium (RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin G, 100 µg/mL streptomycin, 25 mmol/L HEPES, and 0.3 g/L L-glutamine. The viability of mononuclear cells was confirmed by the trypan blue exclusion test. The viable mononuclear cell numbers were counted with a hemocytometer. In several experiments, differential cell counts were obtained from smears of the mononuclear cell fraction treated with Wright’s stain. The viability of mononuclear cells was routinely more than 95%.
Culture of mononuclear cells
After harvesting, the mononuclear cells were rapidly plated onto
a 35-mm tissue culture dish (Falcon 3001) at a concentration of 3
x 106 cells/well and were incubated at 37 C incubator with
5% CO2 for 15–18 h. The culture medium used was described
above. The cells were treated with human GnRH
(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2;
Sigma Chemical Co., St. Louis, MO;
10-5–10-11 mol/L), GnRH-a
([D-Ala6]-LH-RH; Sigma Chemical Co.; 10-6–10-11 mol/L), or
GnRH-ant (antide;
acetyl-ß-[2-naphthyl]-D-Ala-D-p-chloro-Phe-ß-[3-pyridyl]-D-Ala-Ser-N
-[nicotinoyl]-Lys-N-[nicotinoyl]-D-Lys-Leu-N-[isopropyl]-Lys-Pro-D-Ala-NH2; Sigma Chemical Co.;
10-6 mol/L) for the scheduled time interval. For cultures
treated with GnRH, only a single dose of GnRH was added at the
initiation of the experiment. For cultures involving GnRH-a and
GnRH-ant, repeat doses of the analogs were added at 12-h intervals.
RNA extraction and RT-PCR
After incubation, cells were collected, and total RNA was
determined using the RNaid Kit (Bio/Can Scientific, Mississauga,
Canada). The integrity of the RNA sample was verified by
electrophoresis in a formaldehyde-denaturing gel, by spectrophotometric
analysis of the absorbance ratio at 260 nm/280 nm (DU-64
Spectrophotometer, Beckman Coulter, Inc., Hialeah, FL),
and by examining the RT-PCR product of ß-actin mRNA. One microgram of
total RNA, as estimated by spectrophotometry, was subjected to first
strand cDNA synthesis in a 15-µL reaction mixture using an
oligo(deoxythymidine)12–18 primer following the
manufacturer’s instruction (First Strand cDNA Synthesis kit,
Pharmacia Biotech, Morgan, Canada). One microliter of the
15-µL first strand cDNA reaction was used as the template for PCR.
Two pairs of primers were used for examining GnRHR, one pair for the
GnRH and one pair for the IL-2R -subunit (IL-2R; Table 1
). According to their locations in human
GnRHR cDNA (2, 4) (Fig. 1), the primer
pair GnRHR F1/R1 was used to amplify a sequence within the coding
region of GnRHR cDNA spanning the last 202 bp of exon 1 and the first
145 bp of exon 2 (Fig. 1A). The GnRHR F2/R2 primer set amplified
another segment spanning the last 67 bp of exon 1, 220 bp of exon 2,
and the first 10 bp of exon 3 (Fig. 1A). The spanning of two or three
exons in these primer sets excludes the possibility of amplifying
genomic DNA contamination during RNA extraction. The locations of the
primers for examining GnRH mRNA, GnRH F1/R1 primers, are shown in Fig. 1B. These primers have been used to amplify the major portion of the
cDNA encoding the precursor protein for the hypothalamic GnRH and PRL
release-inhibiting factor (GnRH-PIF) (26). It amplifies the coding
region of the signal peptide, the GnRH peptide, and the PIF plus part
of the 3'-untranslated region (Fig. 1B). As an internal reference, we
have also amplified the ß-actin mRNA from the same samples. In a
50-µL reaction mixture, PCR was performed in the presence of 20
mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 2.5 U
Taq DNA polymerase (Life Technologies), 0.2
mmol/L deoxy-NTPs, 0.5 µmol/L primers, and 1–2 µL first strand
cDNA template. Several preliminary studies were performed to validate
the conditions of PCR. It was found that when 1 µg total RNA was used
for RT, subsequent GnRHR mRNA amplification resulted in a progressive
increase in signal intensity up to 37 cycles. Similarly, the signals
for the amplification of GnRH, IL-2R, and ß-actin were linear up
to 36, 32, and 26 cycles, respectively. Therefore, 23–35 cycles of PCR
were performed according to the mRNA to be amplified. The PCR was
carried out in a Perkin-Elmer/Cetus DNA Thermal Cycler
(Palo Alto, CA) in the following sequence: denaturation at 94 C for 1
min, annealing at 56–64 C for 40 s, extension at 72 C for 1 min
30 s, and a final extension for 15 min at 72 C after the last
cycle. PCR products (20 mL) were subsequently subjected to 0.9–1.5%
agarose gel electrophoresis and stained with ethidium bromide. Several
positive and negative controls were used in the PCR. Human
granulosa-luteal cells were used as positive controls (10, 11, 27). A
RT reaction was carried out without the addition of reverse
transcriptase, and the resulting product was subjected to PCR to
exclude the possibility of genomic DNA contamination. Secondly, PCR was
performed without the presence of template DNA to test for
cross-contamination of samples. To compare the mRNA levels under
regulation, semiquantitation of the PCR product was obtained by
concomitant examination of and comparison with the ß-actin control in
the same sample.
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The PCR products were transferred to a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL) and hybridized with digoxigenin (DIG)-labeled cDNA probes that had been prepared by a random hexanucleotide-primed synthesis according to the protocol from the DIG DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN). Briefly, the nylon membranes were hybridized with the DIG-labeled probes overnight at 42 C in a solution containing 50% formamide, 5 x SSC (standard saline citrate), 0.1% (wt/vol) N-lauroylsarcosine, 0.02% (wt/vol) SDS, and 2% blocking reagent. The nylon membranes were then washed twice with 2 x SSC-0.1% SDS at room temperature for 5 min each time and twice with 0.1 x SSC-0.1% SDS at 68 C for 15 min each. The hybridization signals were detected by an enzyme immunoassay according to the DIG Luminescent Detection Kit (Boehringer Mannheim). Briefly, the hybridized probes were detected with anti-DIG Fab fragments conjugated to alkaline phosphatase and were then visualized with the chemiluminescence substrate CSPD. Enzymatic dephosphorylation of CSPD by alkaline phosphatase leads to a light emission that can be recorded on x-ray film. The signal intensity was quantified by a video densitometer (model 620, Bio-Rad Laboratories, Inc., Richmond, CA).
Cloning and sequencing of GnRHR and GnRH cDNA
The PCR products obtained from primer sets GnRHR F1/R1 and GnRH FR(R) were subcloned into a TA PCR 2.1 vector (Invitrogen, San Diego, CA). Three clones each were subsequently analyzed by the dideoxynucleotide chain termination method using either the T7 sequencing kit (Pharmacia Biotech) or the ABI Prism model 377 Automated DNA Sequencer. The sequencing results were matched with the available sequences using the BLAST (basic length alignment search tool) program provided by the National Center for Biotechnology Information.
Statistical analysis
The data obtained in this study were analyzed with one-way ANOVA and post-hoc comparisons with the Bonferroni test or the Student-Newman-Keuls test. P < 0.05 was considered statistically significant.
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Results |
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RT-PCR coupled to Southern hybridization of total RNA obtained
from mononuclear cells confirmed that the GnRHR and GnRH genes are
expressed in human peripheral blood lymphocytes (Fig. 2).
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). The primer set GnRHR F1/R1 includes an upstream GnRHR F1 primer
that is located in exon 1 and a downstream primer GnRHR R1 that is
located in exon 2, which amplifies a 347-bp fragment from mononuclear
cells (Fig. 2A). This DNA was subsequently found to have the same
sequence as the published human pituitary GnRHR cDNA sequence (2, 13),
and its identity was confirmed by Southern hybridization. Human ovarian
granulosa-luteal cells also expressed this signal (Fig. 2A). An
approximately 4.2-kb intron sequence between the two exons (exons 1 and
2) ensured that the amplified PCR product originated from the GnRHR
mRNA rather than from the genomic DNA. This result was further
confirmed by the use of the GnRHR F2/R2 primer set. The GnRHR F2 is
located at exon 1, and the GnRHR R2 is located at the junction of exons
2 and 3 (crossing intron 2). A 297-bp PCR product was detected (Fig. 2A, L3 column) and was found to have the same sequence as the pituitary
GnRHR cDNA. This primer set spans two introns (intron 1, 4.2 kb; intron
2, 5 kb) (7), and this further confirms the mRNA origin of
amplification. None of the negative controls (RT without reverse
transcriptase and PCR without template) showed visible signal, and this
essentially excluded the possibilities of genomic DNA contamination and
cross-contamination of samples.
The primers GnRH F1/R1 were used to detect of the presence of GnRH mRNA
in mononuclear cells (Table 1), and a 380-bp PCR product was
identified. This product has the same size as that found in
granulosa-luteal cell (Fig. 2B), which was identified as GnRH
hypothalamic GnRH cDNA sequence (26), and its authenticity was
confirmed by Southern blot hybridization and DNA sequence analysis.
Similarly, none of the negative controls showed any hybridization
signal.
Homologous regulation of GnRHR, GnRH, and IL-2R mRNA by GnRH and GnRH-a
During in vitro cultures, different doses of GnRH were
added to the medium, and the mononuclear cells were cultured for
24 h, at which time they were harvested. Total RNA was extracted
for subsequent RT-PCR using the primer sets listed in Table 1. GnRHR,
GnRH, and IL-2R mRNA were semiquantitated using ß-actin mRNA as an
internal control (Figs. 3-6). It was
found that GnRH increased the expression of GnRHR mRNA in a
dose-dependent manner, with a maximal increase of 158 ± 16%
(mean ± SEM) of control at 10-8 mol/L
GnRH (P < 0.05; Fig. 3, A and B). This stimulatory
effect was antagonized by the concomitant addition of 10-6
mol/L GnRH-ant to the culture medium (Fig. 3C). In contrast,
experiments using GnRH-a at 10-6-10-11 mol/L
did not change the mRNA levels (Fig. 3D). According to the results of
dose-dependent studies, a time-course study of the effect of GnRH on
mononuclear cells was performed by culturing the cells either with or
without 10-8 mol/L GnRH. At 3, 6, 12, and 24 h, the
cells were collected for study. It was found that GnRHR mRNA increased
gradually after in vitro culture, and this increase was
further augmented by treatment with GnRH for up to 24 h (Fig. 4). This augmented increase in GnRHR mRNA
was statistically significant after 12 h (P <
0.05 at 12 and 24 h).
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subunit gene was highly expressed in
mononuclear cells. At 24 h, its baseline mRNA was stimulated by
both GnRH and GnRH-a (Fig. 6). GnRH stimulated IL-2R mRNA
expression significantly in a dose-dependent manner up to approximately
150% of the control value at 10-5-10-6 mol/L
at 24 h (Fig. 6, A and B).
Similarly, GnRH-a stimulated this expression, but only significantly at
the 10-10-10-11 mol/L level (Fig. 6C).
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Truncated GnRHR cDNA was produced by RT-PCR by the use of primer set GnRHR F1/R1; it consisted of 347 bp, was subsequently sequenced, and was found to be identical to the published sequence of pituitary GnRHR cDNA (4, 13). It was thus confirmed that the GnRHR transcript expressed in mononuclear cells has an identical nucleotide sequence as the pituitary GnRHR cDNA, at least within this 347 bp. This sequence composes approximately 35% of a complete pituitary GnRHR cDNA-coding region. Subsequently, the full-length cDNA for GnRHR from mononuclear cells was cloned by means of PCR. The primers used were as follows: sense primer, 5'-GAA AAT ATG GCA AAC AGT GCC TCT-3'; and antisense primer, 5'-ATC AAT CAC AGA GAA AAA TAT CCA-3'. The size of the amplified PCR product was 998 bp. After PCR, the product was cloned into pUC19 vector by blunt end ligation. The cDNA sequence from the mononuclear cells was identical to the published sequence for pituitary GnRHR.
Similarly, the 380-bp cDNA by RT-PCR encoding the GnRH was sequenced. The GnRH transcripts in mononuclear cells were identical to the hypothalamic GnRH-PIF cDNA, at least within this 380-bp fragment, those from 34 bp downstream of start codon ATG to 134 downstream from the stop codon TAA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA expression at 24 h. Recent evidence suggests that GnRH-binding sites are also present in extrapituitary tissues, such as the ovary, testis, prostate, breast, spleen, and thymus, and in lymphocytes (2, 9, 10, 11, 13) and lymphoid tissues (14, 15, 16, 18, 27). There was no prior report describing GnRHR gene expression at the transcriptional level, except one study showing that GnRHR mRNA cannot be amplified from human mononuclear cells (12). The present study shows that GnRHR is expressed in mononuclear cells, and the identity of this transcript is confirmed by DNA sequence analysis. The results in this study showed that in addition to its expression in mononuclear cells, GnRHR mRNA is homologously regulated, although there may be certain differences compared to the regulation of pituitary GnRHR mRNA (28, 29, 30, 31, 32). In conjunction with the observation of positive effects of GnRH on cell surface GnRHR (15, 21, 28, 33, 34, 35, 36, 37), these results suggest that GnRHR may play an active role in regulating lymphocyte function. However, the present study cannot identify exactly which regulating lymphocyte subpopulation(s) contains this mRNA. Future studies are warranted to answer this question.
In this study, we observed that there is difference between the effects of GnRH and GnRH-a on the steady state GnRHR mRNA level. This difference may be explained by the fundamental differences in the biological activities of GnRH and GnRH-a. It was reported that native GnRH has a relatively short half-life of 87 min in medium 199 with 10% horse serum (28). Analogs of GnRH, however, have been designed to resist degradation and to increase biological potency. Therefore, most GnRH analogs have potencies 20- to 200-fold greater than the native or synthetic GnRH and have much longer half-lives both in in vivo and in vitro. The difference between GnRH and GnRH-a in this study may thus be explained by the relative potencies and half-lives of these two agents in culture. Due to its short half-life and lower potency, the use of single doses of GnRH in culture mimics the action of a single pulse of GnRH, thus bypassing the possible desensitization of the GnRHR (and probably GnRHR mRNA).
The clinical significance of GnRH mRNA expression in lymphocytes is not clear. Previously, it has been known that the GnRH concentration is low in the human portal system (16) and is further diluted and metabolized in the general circulation. This makes it unlikely that hypothalamic GnRH could exert a significant action on extrapituitary sites, including peripheral lymphocytes. This suggests that an alternative source of endogenous GnRH may play a physiological role in extrapituitary sites in an autocrine or paracrine manner. For example, human placenta has been shown to express GnRH transcript identical to that in the hypothalamus (GnRH-PIF) (26, 38). The presence of GnRH-PIF mRNA in lymphocytes with a nucleotide sequence identical to that in the hypothalamus is consistent with the genomic analysis that only a single GnRH gene is present in the human (26). In addition, this result indicates that lymphocytes may secrete an authentic GnRH that is able to act on both lymphocytes themselves and on other tissues or cells. For example, GnRH has been demonstrated to regulate folliculogenesis and steroidogenesis in the ovary (39). It is therefore speculated that resident ovarian lymphocytes may play a role in these regulations through the production of GnRH.
Due to the presence of GnRHR and GnRH gene expressions in lymphocytes,
the potential direct effect of GnRH on the human lymphocyte was also
assessed, and it was found that GnRH and GnRH-a stimulated IL-2R
chain mRNA. IL-2 plays a pivotal role in the activation and
proliferation of lymphocytes through a receptor complex (IL-2R)
composed of at least of three distinct chains, i.e. 
-,
ß-, and -chains (24, 40). The noncovalent combination of these
three chains constitutes the high affinity receptor with a
Kd at the 10-11 mol/L level. There has been a
report showing that GnRH induction of rat thymus lymphocyte activation
in vitro is accompanied by a specific increase in the
IL-R-positive T cell (15). In in vivo studies, we have
shown that the proportion of CD25-positive (IL-2R-positive) T cell in
peripheral mononuclear cells is down-regulated after 2-week treatment
with GnRH-a (41, 42). However, IL-2R itself is not able to process
lymphocyte activation and proliferation due to the lack of a
cytoplasmic domain, which is needed for signal transduction (40). In
this study, we chose IL-2R mRNA as another indirect indicator of
lymphocyte activation. We observed that IL-2R mRNA is stimulated by
relatively short term (24-h) GnRH and GnRH-a stimulation. This may
indicate that either endogenous or exogenous GnRH or GnRH-a may be able
to modulate the expression of the IL-2R gene, which subsequently may
affect lymphocyte activation and proliferation. However, the clinical
significance of this short term effect on the lymphocyte function is
not yet clear. It is expected that a long term in vivo study
will be able to address this question.
In summary, the present study indicates that GnRH and GnRHR mRNAs are
expressed in human peripheral lymphocytes and these mRNAs are identical
to their pituitary and hypothalamus counterparts, at least in the major
portion of the coding regions. The importance of GnRH as a
physiological regulator of these mRNAs was also demonstrated by an
increase in GnRHR mRNA and a decrease in GnRH mRNA after short term
culture with GnRH. In addition, GnRH and GnRH-a are also shown to
regulate IL-2R mRNA expression; this suggests an important role of
GnRH in lymphocyte function. The above results thus identified a close
interaction between the immune and endocrine systems through certain
mediators, including the classical neuropeptide hormone, GnRH, and its
receptor. Because of the active roles that lymphocytes play in most if
not all aspects of human physiology, it is speculated that lymphocytes
may participate in various human reproductive functions through the
secretion of GnRH and through the action of the latter on its own GnRHR
or on other cells in an autocrine or paracrine manner. In addition, the
exogenous administration of GnRH and its analogs (GnRH-a and GnRH-ant),
as is common in clinical practice, may theoretically exert a certain
impact on the immune system.
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Footnotes |
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Received May 14, 1998.
Revised September 23, 1998.
Accepted October 20, 1998.
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K. W. Cheng, P. S. Nathwani, and P. C. K. Leung Regulation of Human Gonadotropin-Releasing Hormone Receptor Gene Expression in Placental Cells Endocrinology, July 1, 2000; 141(7): 2340 - 2349. [Abstract] [Full Text] [PDF]
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J. D. Jacobson, M. A. Ansari, M. Kinealy, and V. Muthukrishnan Gender-Specific Exacerbation of Murine Lupus by Gonadotropin-Releasing Hormone: Potential Role of G{alpha}q/11 Endocrinology, August 1, 1999; 140(8): 3429 - 3437. [Abstract] [Full Text]
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