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q/111
Section of Endocrinology, Children’s Mercy Hospital, University of Missouri-Kansas City School of Medicine and the School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64108
Address all correspondence and requests for reprints to: Jill D. Jacobson, M.D., Section of Endocrinology, Children’s Mercy Hospital, 2401 Gillham Road, Kansas City, Missouri 64108. E-mail: jjacobson{at}cmh.edu
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Abstract |
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q/11. Competitive RT-PCR revealed that GnRH
administration was associated with increases in the expression of
Gq/11 mRNA, compared with vehicle, in spleens in
ovariectomized females but not in castrated males. Immunoblot analysis
revealed a similar pattern. We conclude that gender differences in
expression of Gq/11 may contribute to gender differences
in immunity and/or autoimmune disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One hypothalamic hormone with immunomodulatory properties is GnRH (also known as LHRH). Numerous in vivo and in vitro studies have demonstrated that GnRH exerts predominantly stimulatory actions on both B and T lymphocyte function (3, 4). Moreover, immune cells have been shown to produce GnRH messenger RNA (mRNA) and peptide (5, 6). We have previously reported that GnRH analogues modulate the expression of murine lupus independently of effects on gonadal steroids (7). We now speculate that GnRH may play a key role in the gender differences in immunity and/or autoimmunity. We note the following clinical and experimental observations: 1) GnRH has been shown to be immunostimulatory; 2) it is produced by lymphocytes; 3) its production and action are positively regulated by estrogens; 4) its action is negatively regulated by androgens; 5) GnRH action is high in women during reproductive years, when autoimmune diseases occur; and 6) gonadal failure is associated with both a loss of negative feedback on GnRH and with a high incidence of autoimmune disorders (8, 9, 10).
In this report, we demonstrate that gonadectomized male and female mice
respond to the administration of native GnRH with exacerbation of
disease severity in a sexually dimorphic fashion. Because exposure to
androgens and estrogens is known to alter GnRH receptor expression at
the level of the pituitary (11, 12, 13, 14, 15, 16), we speculated that gender
differences in GnRH receptor expression might be demonstrable in immune
cells. We measured expression of GnRH receptor mRNA in the thymus and
spleen of gonadectomized mice, by competitive RT-PCR. Because GnRH
administration has been associated with lymphocyte activation and
increased expression of the interleukin-2 (IL-2) receptor (17), we next
measured IL-2 receptor mRNA expression, by competitive RT-PCR, in
lymphoid organs in males vs. females. Because GnRH is known
to exert actions through G proteins, specifically through two highly
homologous G proteins Gq and G11
(hereafter referred to together as Gq/11) (18, 19), we
sought gender differences in expression of Gq/11 mRNA
and protein in lymphoid tissue.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experimental design
We expected that IL-2 receptor expression would be an early
event in the course of disease. We therefore performed RT-PCR for IL-2
receptor and flow cytometric analysis of the IL-2 receptor at an early
time point (2 weeks). We expected that changes in GnRH receptor, IL-2
receptor, and Gq/11 would precede any alterations in
disease severity. Previous studies have shown dynamic alterations in
GnRH receptor and GnRH responsiveness at the level of the pituitary
after gonadectomy in both male and female rats (23, 24). It seems that
these alterations begin to plateau by 2–3 weeks post surgery (23, 24).
We postulated that similar effects would be seen in murine immune
cells. We therefore used 2 weeks as our initial time point for
molecular studies of all genes. RT-PCRs for GnRH receptor, IL-2
receptor, and Gq/11 were repeated after six weeks of
treatment. Autoantibody levels and hematuria were measured every 6
weeks. Serum IgG levels were measured at 12-week intervals. Serum
gonadal steroids were measured once, at 12 weeks. Refer to Table 1
for design of the study.
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Injections
GnRH (native decapeptide) was purchased from Sigma Chemical Co. (St. Louis, MO). Animals were injected sc in the
nape of the neck six times weekly, with 100 µg GnRH or 100 µl
vehicle consisting of 0.9 N saline.
Hormone measurements
Serum estradiol and testosterone were measured by RIA using
commercial kits (Coat-A-Count, Diagnostic Products,
Los Angeles, CA). The lower limit of detection for serum estradiol was
8 pg/ml. The lower limit of detection for testosterone was 4 ng/dl.
Serum LH and PRL were measured by RIA using previously described
methods (25, 26).
Sera
Sera were collected from blood obtained at 6 weeks by
retroorbital puncture after isoflorane anesthesia.
Clinical tests
Total IgG concentrations were measured by single radial
immunodiffusion assay using immunodiffusion plates containing
monospecific antiserum for IgG (ICN Biomedicals, Inc.,
Costa Mesa, CA). Anti-DNA antibody levels were measured by a previously
described enzyme-linked immunosorbent assay technique (27). Sera for
anti-DNA antibody measurements were stored at -20 C. All samples from
each time point were run in the same assay in an effort to avoid
interassay variability. Urine was tested for blood by urinalysis
reagent strips (Miles, Inc., Elkhart IN). Hematuria was scored blindly
as follows: negative = 0; trace = 1; small = 2;
moderate = 3; large (gross) = 4.
RNA purification
RNA was isolated by a the technique of Chomczynski, using
TRI Reagent (Molecular Research Center, Inc.,
Cincinnati, OH), as follows (28): whole organs were individually
removed, placed in 1 ml TRI reagent, homogenized, and placed on
dry ice. RNA was extracted with chloroform, centrifuged, precipitated
with isopropanol, recentrifuged, washed in 75% ethanol, and air dried
for 1 h. The pellet was resolubilized in 25–200 µl of 0.05%
diethylpyrocarbonate-treated H2O. RNA concentration was
determined by optical density readings at 260 and 280 nm. Quantity of
RNA was further assessed by measurement of ß-actin by competitive
RT-PCR.
RT
RNA (5 µg) from each individual organ was transcribed into
complementary DNA (cDNA) in a total vol of 50 µl using random
hexamers to prime the reaction (Perkin-Elmer-Cetus,
Norwalk, CT) and mouse murine leukemia virus reverse transcriptase
(Life Technologies, Inc., Gaithersburg, MD). Control
reaction tubes were identical except for the omission of reverse
transcriptase in one set and RNA in another set (29).
Primer design
We used the published sequence of the murine GnRH receptor mRNA
(30) and our previously published primer sequences to amplify GnRH
receptor mRNA (31). Using the published sequence of the homologous
genes Gq and G11 (Gq/11)
(32), we have synthesized the following oligonucleotide primers to
amplify Gq/11 mRNA: sense: 5'-GGA GGT CGA TGT GGA
GAA GG-3' antisense: 5'-CTG CCC GCC CAC ATC CAC
CA-3'. We used commercially available primers for PCR of murine
IL-2 receptor and ß-actin (CLONTECH Laboratories, Inc.,
Palo Alto, CA). These primers also flank intron-exon boundaries.
Competitor construction
Because quantitation is of importance, we used a competitive PCR
technique for all genes. We used mimic construction kits
(CLONTECH Laboratories, Inc.) to construct nonhomologous
DNA fragments flanked by the primer templates for all genes (33). The
competitive fragment corresponding to the GnRH receptor is 323 bp in
length and is easily distinguishable from the gene product band (198
bp). The fragment corresponding to ß-actin is 344 bp in length and is
easily distinguishable from the gene product band (540 bp). The
fragment corresponding to the IL-2 receptor is 448 bp in length and is
easily distinguishable from the gene product band (700 bp). The
fragment corresponding to Gq/11 is 554 bp in length and
is easily distinguishable from the gene product band (605 bp). Primers
were used to amplify both the target genes and their corresponding
nonhomologous DNA fragments in the same reaction tubes.
Competitor titration
To optimize the working concentration of each competitor, a
standard curve was produced by amplifying serial 10-fold dilutions of
competitor in the presence of a constant amount of cDNA from a positive
sample. The log ratio of target:competitor was plotted against the log
of femtomoles of competitor. We have previously demonstrated a linear
relationship between the log ratio target:competitor vs. log
competitor using our primers for the GnRH receptor (31). A linear
relationship was observed between the log ratio of target to competitor
in the range of 10-6 fmol to 10-2 fmol
for GnRH receptor (r2 = 0.951; slope = -1.34). A
linear relationship was observed between the log of the ratio of target
to competitor in the range of 102–107 fmol for
ß-actin (r2 = 0.974; slope = -0.709). A linear
relationship was observed between the log of the ratio of target to
competitor in the range of 10-3–10-0
fmol for the IL-2 receptor (r2 = 0.956;
slope = 0.609) and from 10-1–104 fmol
for Gq/11 (r2 = 0.971; slope =
-1.15). Competitor was added to the samples within these linear
ranges.
Amplification efficiency
To establish that the target cDNA and competitor cDNA were
amplified at similar efficiencies, the log density of target DNA and
competitor cDNA were plotted against cycle number. For each gene, the
linear portions of the two curves exhibited very similar slopes,
indicating that the amplification efficiencies of the target DNA and
competitor DNA were equal, as previously demonstrated for GnRH receptor
(31).
PCR
PCR was performed under intermediate stringency, by mixing 5
µl of the cDNA with PCR buffer, 2 mM MgCl2,
0.2 mM deoxynucleotide triphosphates, 2ng/ml sense and
antisense primers, 5 U Taq polymerase, 2 µl mimic and 27
µl ribonuclease- and deoxyribonuclease-free H2O in a
total vol of 50 µl. For radiolabeled PCR, 0.5 µl 32P
labeled deoxycycidine triphosphate was added to the reaction mixture.
PCR cycles were programmed as follows: 95 C for 2 min; 1 cycle: 95 C
for 1 min, 60 C or 65 C for 1 min; 35 cycles: 72 C for 7 min; 1 cycle:
4 C soak (31).
Electrophoresis
Aliquots (5 µl) of PCR reactions were subjected to
electrophoresis through 5% polyacrylamide gels or 1.8% agarose gels.
Product and competitor bands were quantitated using densitometry
(Molecular Dynamics, Inc., Sunnyvale, CA). Data are
expressed as ratio of product:competitor, which correlates linearly
with the amount of competitor added for each gene.
Mononuclear cell preparation
Peripheral blood mononuclear cells (PBMC) and splenic
mononuclear cells (SMC) were isolated from heparinized blood by
centrifugation over Ficoll-Hypaque gradients (Sigma Chemical Co.).
Mononuclear cell subset analysis
Phenotypic analysis of PBMC and SMC was performed by
immunofluorescent staining and flow cytometry, as previously described
(7). PBMC and SMC were labeled with biotin-conjugated anti-CD25,
(PharMingen, San Diego, CA) or isotype-matched control
antibodies. For flow-cytometric analyses, 0.5–1 x
106 cells were incubated on ice for 30 min with 5 µg/ml
monoclonal antibody in 30 µl PBS/1% FCS/0.1% NaN3.
Cells were washed twice in cold PBS/FCS/NaN3, and
streptavidin phycoerythrin was added in a second incubation in 20 µl
PBS with 0.2% NaN3 at 4 C for 20 min. After washing,
10,000 cells were analyzed on an Epics Profile II flow cytometer
(Beckman Coulter, Miami, FL). Gates were
established on viable cells using forward and side scatter parameters.
Only viable cells were analyzed.
Iodination of buserelin
The labeling was carried out for 2 min using 2.5 µg of the
peptide buserelin [D-Ser(TBU6)des-Gly10,
LHRH-N-ethylamide (Hoeschst AG, Frankfurt,
Germany)] in 30 µl 0.5 M sodium phosphate (pH 7.2), 500
µCi carrier-free Na125I, and 600 ng chloramine-T in 10
µl of 0.5 M sodium phosphate (pH 7.2), as previously
described in full detail (3, 34). The reaction was stopped by the
addition of 1 ml eluant buffer (10 mM Tris-HCl/0.1% BSA
0.1% NaN3, pH 7.6, at 25 C) and immediately layered onto a
1 x 10 cm column of Sephadex G-25 (Sigma Chemical Co.). Specific activity (280–380 µCi/µg) was determined by
equating the ED50 value (in µCi) of a saturation curve
with ED50 value (in µg) of a displacement curve obtained
in the same assay (3).
GnRH receptor binding studies
GnRH receptor assay was carried out as previously described (3),
in a total vol of 500 µl, for 90 min at 0–4 C. Assays were performed
in triplicate in polypropylene test tubes, presoaked overnight in 1%
albumin. The tubes contained 200 µl buffer (40 mM
Tris-HCL, 0.1% BSA, pH 7.6), 100 µl [125I] buserelin
(
150,000–180,000 cpm), 100 µl unlabeled buserelin (10 µg/tube)
or buffer. Bound hormone was separated after washing with 3 ml ice-cold
Tris-HCL buffer, pH 7.6, and by centrifugation at 15,000 x
g. The supernatants were aspirated, and the radioactivity
was contained in the pellet counted in a liquid scintillation counter
(Packard Instruments Co., Downers Grove, IL) at 70% efficiency.
Immunoblot analysis
G protein expression was determined by immunoblot analysis, as
previously described (35). Membrane proteins were solubilized and
resolved by electrophoresis. The resolved proteins were
electrophoretically transferred to nitrocellulose membrane (Sigma Chemical Co.). The blots were incubated with primary antibody,
rabbit antimouse Gq/11 (1:1000 dilution, 0.05 µg/ml;
Santa Cruz Laboratories, Santa Cruz, CA) for 3 h. Blots were then
incubated with secondary antibody conjugated with alkaline phosphatase
(1:100 dilution, 0.5 µg/ml) for 2 h. The blots were subsequently
washed and then developed by adding the substrate
(5-bromo-4-chloro-3-indolyl phosphate, (Sigma Chemical Co.) and incubating overnight at room temperature with gentle
shaking.
Statistics
Anti-DNA antibody measurements, serum Ig measurements, and
densitometric data were analyzed using a one-way ANOVA and further
analyzed by Student’s t test. Those densitometric data that
were skewed and hematuria data were analyzed by the nonparametric
Kruskal-Wallis one-way ANOVA and further analyzed using the Wilcoxon
rank sum test. Graphic data are expressed as mean ±
SEM of all experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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shows total serum IgG levels in
females and males after 12 weeks of administration of GnRH or vehicle.
Again, GnRH agonist significantly increased disease severity, as
evidenced by IgG levels in females. (P < 0.000001
agonist vs. vehicle treatments in females). No effect of
GnRH agonist on IgG levels was observed in males. Similar results were
observed after 24 weeks of treatment (data not shown).
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shows the later, more severe hematuria
scores in females and males after 30 weeks of administration of GnRH or
vehicle. Again, GnRH agonist significantly increased disease severity,
as evidenced by hematuria levels in females (P < 0.05
agonist vs. vehicle treatment). No effect of GnRH agonist on
IgG levels was observed in males (n = 9–13/group).
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Expression of GnRH receptor mRNA
The observation that GnRH exacerbated disease in females only led
us to the initial speculation that females expressed more GnRH receptor
than males. We measured GnRH receptor mRNA, by competitive RT-PCR,
after treatment with GnRH or vehicle. GnRH agonist treatment was
associated with significantly decreased expression of the GnRH
receptor, compared with vehicle, in spleen in females. The effects were
evident at the 2-week time point (P < 0.005). GnRH
agonist administration exerted no demonstrable effect on expression of
the GnRH receptor in males (Fig. 4). A
similar pattern was observed in the thymus, although the differences
were not statistically significantly different. Similar patterns of
mRNA expression were observed at the 6-week time point (data not
shown).
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shows the results of binding studies
after 2 weeks of treatment.
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). Similar results were
observed in the thymus [ratio of product:competitor in female vehicle
treated vs. female GnRH treated: 0.79 ± 0.04
vs. 1.01 ± 0.06, respectively (P <
0.05)]. GnRH agonist administration did not alter IL-2 receptor mRNA
expression in the male thymus (ratio of product:competitor in male
vehicle treated 0.74 ± 0.07 vs. male GnRH treated
0.66 ± 0.06.) When amplifying the IL-2 receptor, we noted the
presence of weak bands between the target and competitor at high cycle
numbers. The source of the faint bands is unknown. If they result from
amplification of alternate templates, they should not interfere with
coamplification of target and competitor. If they result from
heteroduplex formation between target and competitor, they could affect
the target:competitor ratio. This did not occur, given that the ratio
remained constant throughout multiple cycle numbers.
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). Similar results were
observed in PBMC: mean fluorescent intensity 1.61 ± 0.09 in
female vehicle vs. 2.20 ± 0.15 in female GnRH
(P < 0.01). GnRH administration was associated with a
decrease in mean fluorescence intensity in male PBMC [mean fluorescent
intensity 1.54± 0.11 in male vehicle vs. 1.22 ± 0.07
in male GnRH-treated mice (P < 0.05)].
Expression of Gq/11 mRNA
The observation that the GnRH receptor was down-regulated in
females led us to speculate about postreceptor differences. GnRH is
known to work through G proteins. It is known to exert actions through
Gq/11. We measured Gq/11 mRNA by
competitive RT-PCR after administration of GnRH or vehicle. Females
expressed significantly more Gq/11 mRNA than males
(P < 0.05). GnRH agonist administration significantly
increased expression of the Gq/11, compared with
vehicle, in spleen in females (P < 0.01). GnRH agonist
administration exerted no demonstrable effect on expression of the
Gq/11 mRNA in males (Fig. 6). The effects were apparent at the
2-week time point. Similar results were observed at the 6-week time
point.
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q/11 proteinq/11 protein. No band was visible in
spleens of vehicle-treated males or females. A faint band can be seen
in GnRH-treated males. A representative immunoblot is shown in Fig. 7.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our initial murine studies demonstrated that antagonists to GnRH ameliorated disease severity in (SWR x NZB) F1 hybrid mice, a lupus-prone mouse strain. Results were similar in female mice whether they were intact, gonadectomized, or treated with high-dose estradiol, demonstrating that the effects of GnRH antagonists were independent of estradiol. On the other hand, GnRH agonist administration exacerbated lupus in ovariectomized females (7).
In contrast to our past study using GnRH antagonists, the present study demonstrates that GnRH agonists modulate the severity of murine lupus in a gender-specific fashion. Whereas females respond to GnRH agonist with an exacerbation of lupus, disease severity is unaltered by GnRH administration in males. The results of the present study suggest an additional mechanism for gender differences in expression of autoimmune disease: gender differences in responsiveness to GnRH at the level of the immune system.
Although the present study begins to address the mechanisms for the gender-specific effects of GnRH on murine lupus, we have not attempted to address the importance of gonadotropins. GnRH might act on the immune system directly, by a direct effect on B or T lymphocytes, or indirectly, either by an increase in pituitary gonadotropins or perhaps by an increase in gonadotropin production by immune cells. LH and FSH production and receptor expression by immune cells have been suggested but not confirmed (36, 37). On the other hand, numerous studies have demonstrated the expression of GnRH and GnRH receptor mRNA and protein in immune cells (5, 6, 31, 38, 39). We have previously shown that GnRH mRNA and GnRH receptor mRNA are cyclically expressed in parallel in immune cells during the mouse estrous cycle. This finding lends support for a direct autocrine or paracrine action for GnRH in the immune system (31).
Females display increases in autoantibody and serum IgG levels at the earliest time points tested, reflecting increased disease severity. At the later time points in females, a reduction in anti-DNA antibody levels is seen. This could be attributable to tachyphylaxis to GnRH agonist. GnRH is known to down-regulate its own receptor whenever it is administered in a nonpulsatile fashion (40). A trend toward early mortality was noted in the GnRH-treated females, compared with vehicle-treated females. A negative selection of the most GnRH-responsive females may have contributed to the later waning of autoantibody levels in females.
Our results seem to agree with (and perhaps provide a unifying explanation for) some of the seemingly contradictory reports in the literature relating to the immune actions of GnRH. The numerous studies demonstrating immuno-stimulatory properties of GnRH used immune cells exclusively from females (3, 4, 17). A seemingly contradictory study demonstrated that GnRH exerted immunosuppressive effects on lymphocyte proliferation in vitro (41). The latter study used lymphocytes from healthy male volunteers. Our study suggests that the immune actions of GnRH may be gender specific.
The finding that gender differences existed, even in gonadectomized mice, was surprising, because one would have expected that prepubertal gonadectomy would have eliminated gender differences. One possible explanation for the observed gender differences was that the males and females were incompletely gonadectomized and that estrogens were produced by females in sufficient amounts to worsen disease severity and/or that androgens were produced in sufficient quantities to ameliorate disease. This seems unlikely, given that androgen and estrogen levels were undetectable in all treatment groups including vehicle treatment. Furthermore, necropsies revealed no evidence of gonadal tissue.
Previous studies show that prior exposure to estradiol has a major stimulatory influence on GnRH responsiveness in pituitary cultures (21). A study in adult male volunteers shows that pituitary responsiveness to GnRH analogues was controlled by past exposure to testosterone and not by current levels (42). The current study suggests that past exposure to some factor (or factors) determines the effects of GnRH at the level of the immune system, as well. We have recently demonstrated that splenocytes from female mice respond to GnRH in vitro with an increase in T lymphocyte proliferation, whereas splenocytes from males respond with a suppression (43). Thus, gender differences in GnRH responsiveness persist in lymphocytes, even after removal from the in vivo hormonal milieu. The differences in in vitro T cell proliferation persist, even when long-term gonadectomized mice are used (unpublished observations). Thus, past exposure to some factor or factors induces persistent gender differences in responsiveness to GnRH.
Although the animals in the current study underwent prepubertal gonadectomy, they would have been exposed to gender divergent hormones antenatally. Perhaps differences in in utero hormonal exposure were sufficient to induce long-lasting immunological changes. Recent studies suggest that it is immature thymocytes that are most responsive to the effects of gonadal steroids (44). Prenatal androgen exposure abolishes the responsiveness to GnRH at the level of the pituitary in sheep (45). Prenatal androgens also abolish estrogen responsiveness of GnRH neurons (46). The HPG/Bm mouse model provides additional evidence that prenatal hormonal exposure may be sufficient to induce lasting gender differences in the immune system, as well. This model has a deletional mutation of the GnRH gene (47). Homozygous GnRH-deficient mice do not enter puberty and are infertile. An early study demonstrated gender differences in thymic size between male and female GnRH-deficient HPG/Bm mice, whose only exposure to gender-disparate androgens, estrogens, or GnRH would have been in utero (48).
In our study, GnRH administration was associated with gender-specific
alterations in mRNA expression of the GnRH receptor, IL-2 receptor, and
Gq/11 after 2 weeks of treatment. These differences
might be attributable to gender differences in response to gonadectomy.
We would have had to include sham-operated animals to test this
hypothesis. Such experiments would have been complicated by the fact
that GnRH and GnRH receptor mRNA levels vary dynamically with the
estrous cycle in lymphoid organs in the intact female mouse (31). We
have recently demonstrated that the expression of Gq/11
also varies with the estrous cycle (unpublished observations). At any
rate, the purpose of the present study was to compare the GnRH-induced
exacerbation of disease between males and females, in the absence of
gender disparate gonadal steroids, and to begin to address the
mechanisms for the dimorphism observed.
We initially hypothesized that the increased exacerbation of disease by GnRH in females might relate to increased GnRH receptor expression. Exposure to estrogens has been shown to increase the expression of the GnRH receptor, at least at the level of the pituitary. Androgens have been shown to decrease GnRH receptor mRNA and protein. Gender differences were observed. Vehicle-treated females displayed increased GnRH binding capacity, compared with vehicle-treated males. With GnRH administration, however, females demonstrated a reduced expression of GnRH receptor mRNA and decreased GnRH binding. GnRH did not alter GnRH receptor expression in males.
Previous studies have shown that GnRH responsiveness in pituitary cells
does not correlate directly with the expression of the GnRH receptor.
Authors of those studies have speculated that postreceptor differences
may explain these observations (11, 12, 24). Our GnRH receptor
expression findings suggested the possibility that gender differences
in immune responsiveness to GnRH might relate to postreceptor
differences in immune cells. We were able to demonstrate that
splenocytes from females expressed more Gq/11 mRNA than
males and that this expression was up-regulated after 2 weeks of
exposure to GnRH. Protein studies supported these findings.
Our observations are consistent with the possibility that gender
differences in responsiveness to GnRH might contribute to gender
differences in the immune system and/or to gender differences in the
expression of autoimmune disease. Gender differences in expression of
the GnRH receptor or of Gq/11 might contribute to gender
differences in responsiveness to GnRH. These differences may relate to
antenatal exposure to estrogens, androgens, and/or GnRH.
Further studies, using knockout models of Gq,
G11, and estrogen receptors, as well as the
GnRH-deficient HPG/Bm mouse strain, will be invaluable in disentangling
evidence questioning which hormonal influences contribute to the gender
differences in responsiveness to GnRH. These models will also provide
valuable clues in developmental neuro-immunoendocrinology.
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Footnotes |
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Received January 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and luteinizing
hormone-â messenger ribonucleic acid (RNA) of male and female
rats after castration: quantitation using an op-timized RNA dot
blot hybridization assay. Endocrinology 119:691–698[Abstract]
–chain
messenger ribonucleic acids that are regulated by GnRH in
vitro. J Clin Endocrinol Metab 84:743–750This article has been cited by other articles:
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