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Division of Endocrinology, Department of Internal Medicine and the Center for Research in Reproduction, University of Virginia, Charlottesville, Virginia 22908
Address all correspondence and requests for reprints to: Alan C. Dalkin, University of Virginia, Department of Internal Medicine, P.O. Box 801387, Charlottesville, Virginia 22908. E-mail: acd6v{at}virginia.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, LHß, and FSHß) is the regulation
of transcription [messenger RNA (mRNA) synthesis]. Gonadotropin
subunit transcription rates increase after gonadectomy and following
exogenous GnRH pulses. However, prior studies of subunit mRNA synthesis
were limited by the available methodology that did not allow
simultaneous measurement of gene transcription and mature mRNA
concentrations. The purpose of the current studies was to: 1) develop a
reliable and sensitive method for assessing transcription rates by
measuring gonadotropin subunit primary transcript RNAs (PT, RNA before
intron splicing); 2) investigate the PT responses to GnRH following
castration or exogenous GnRH pulses; 3) characterize the
half-disappearance time for the three PT species after GnRH withdrawal;
and 4) correlate changes in PT concentration with steady-state
gonadotropin subunit mRNA levels measured in the same pituitary RNA
samples.
Using oligonucleotide primers that flanked intron-exon boundaries,
quantitative RT-PCR assays for each subunit PT species were developed.
These assays require only ng amounts of RNA to measure each
gonadotropin subunit PT and allow us to measure both PTs and
steady-state mRNAs in a single pituitary RNA sample. Primary transcript
concentrations in intact male rats showed a relative abundance of
> LHß 
FSHß, similar to the relationship found
previously for mRNA levels. Additionally, each PT species was only
1–2% as abundant as the corresponding mRNA. One week after
castration, gonadotropin subunit PT levels were increased (: 3-fold,
LHß: 6-fold, and FSHß: 3-fold) in a pattern similar to subunit
mRNAs. Administration of GnRH antagonist to 7-day castrate male rats
resulted in a rapid decline in PT concentrations with a
half-disappearance time of 2.7 h for LHß and 0.8 h for
FSHß, significantly faster than earlier measurements of the
half-disappearance time for mature mRNA. Finally, in a GnRH-deficient
male rat model, LHß and FSHß PT concentrations increased 4- to
6-fold 5 min after a GnRH pulse and then declined toward levels seen in
control animals.
These data indicate that the effects of GnRH on subunit gene transcription are an important determinant of gonadotropin regulation. The appearance and disappearance of PT RNA occurs more rapidly than changes in mature mRNA. Additionally, concentrations are elevated in long term castrates, and following an exogenous GnRH pulse the transcriptional burst is rapid and brief.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit while possessing
distinct ß subunits, the latter conferring biological actions (for
review, see Refs. 1, 2). The - and ß-subunit
messenger RNAs (mRNAs) are encoded by different genes
(3, 4, 5), the expression of which may be regulated in either
a coordinate or differential fashion. Although gonadal steroid and
peptide hormones can exert selective actions on one or more of the
gonadotropin subunit gene products, variations in the GnRH signal
pattern play an important role in the maintenance of normal gonadotrope
function and reproductive capabilities (1, 2). GnRH secretion increases following gonadectomy in both sexes and during the proestrus LH surge in the female rat, coincident with increased expression of the gonadotropin subunit genes (6, 7, 8, 9). Moreover, blockade of GnRH action results in reduced gonadotropin subunit mRNA concentration (10, 11). The parameters of the GnRH signal pattern initiate and maintain specific gonadotrope responses, with alterations in pulse amplitude and/or frequency resulting in selective gene expression. Although GnRH may alter gonadotropin subunit mRNA concentrations via altered mRNA stability (11, 12, 13), GnRH appears to exert its main action by regulating mRNA synthesis (transcription). Previous data from our laboratory has revealed that, similar to mRNA expression, variations in GnRH pulse pattern differentially alter transcriptional responses, suggesting that GnRH action at the level of mRNA synthesis, is of central importance for normal gonadotrope function (14).
To date, measurement of gonadotropin subunit gene transcription has been assessed using the nuclear run-on assay (for review see Ref. 15). This methodology uses isolated nuclear preparations to quantify the rate of mRNA synthesis via incorporation of radiolabeled UTP into newly formed RNA (primary transcript). However, this technique has several limitations for physiologic studies. Specifically, three to four pituitary glands are needed to provide sufficient nuclear material for a single measurement, increasing cost and preventing assay replicates. Also, the isolation of nuclei requires a sucrose cushion separation procedure that makes isolation of cytoplasmic RNA for simultaneous measurement of mRNA expression difficult. Hence, our prior studies of GnRH regulation of gene transcription have been limited to several key physiologic scenarios including the response to varying GnRH pulse frequency and duration of treatment.
Recent reports suggest that the RT-PCR assay can be adapted for measurement of primary transcript (PT) RNA (16). PT is newly formed RNA that includes both exon and intron sequences, before splicing. Thus, PT expression is closely linked to gene transcription and should directly reflect mRNA formation rate. One purpose of the current studies was to develop quantitative RT-PCR assays to measure gonadotropin subunit PT to assess transcription in physiologic studies and avoid many of the limitations noted for run-on assays. The RT-PCR assays require small amounts of RNA and therefore allow for the measurement of all three PT species as well as the three subunit mRNAs from the RNA extracted from a single pituitary. Furthermore, we aimed to use these techniques to: 1) characterize the changes in PT expression after castration as a means to provide a comparison to existing data using the run-on assay; 2) investigate the disappearance rate of PT RNA in castrate male rats following GnRH blockade; and 3) examine the pattern of PT responses to a single GnRH pulse.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Where indicated, we used a GnRH-deficient male rat model. Adult male rats (225–250 g) were castrated and immediately treated with testosterone via sc implants. In these animals, constant serum concentrations of testosterone, average 3.2 ng/ml, suppresses endogenous GnRH secretion (17). To administer GnRH, an indwelling cannula was inserted into the right jugular vein at the time of castration. GnRH was administered at both a physiological amplitude and frequency (25 ng in 0.5 ml 0.9% saline-0.1% BSA, every 30 min) as previously described (14).
For studies to determine primary transcript disappearance, 6-day castrate male rats had right jugular vein catheters inserted and 24 h later (7 days following castration) were treated with the GnRH antagonist LRF-147 (kindly provided by Dr. Jean Rivier, Salk Institute, La Jolla, CA) 30 µg iv every 8 h (11).
RNA preparation, measurement of serum gonadotropins, and
measurement of gonadotropin subunit mRNAs
To quantify both mRNA and PT RNA species in a single pituitary
gland, total cellular RNA was isolated from each pituitary. Total
pituitary RNA was extracted using the acid guanidinium method
(18). Residual genomic DNA was removed by treatment with 1
U RNase Free DNase I/µg RNA (Roche Molecular Biochemicals, Indianapolis, IN) at 37 C for 1 h. RNA
preparations were confirmed to be DNA free by (-) RT-PCR for each
subunit. , LHß, and FSHß mRNA concentrations were determined
using a modification of our dot blot hybridization assays (19, 20). As the pituitary samples cannot contain DNA (to perform
quantitative RT-PCR), we expressed our results per 100 µg pituitary
RNA. As an additional modification of mRNA quantification, we
synthesized sense strand RNA for each gonadotropin subunit from
existing complementary DNA (cDNA) constructs (SP6 or T7 polymerase,
Promega Corp., Madison, WI). For each subunit
assay, the corresponding sense strand RNA was spotted and affixed to
nitrocellulose filters (10, 50, 500, 1000 pg/spot) in duplicate using
standard methods (19, 20). These dots were excised and
counted, the CPMs bound per pg sense strand RNA plotted and a linear
standard curve generated for each filter. Picograms of mRNA
expressed/RNA sample were then calculated using this standard curve.
Results were then converted to molar quantities and final values are
expressed as femtomoles of cDNA bound/100 µg pituitary total RNA.
Construction of the gonadotropin subunit primary transcript
competitive templates
The general scheme for the development of each quantitative
RT-PCR assay for the three gonadotropin PT RNA species was similar.
Initially, oligonucleotide primers were used to PCR amplify a genomic
DNA fragment containing an intron. This DNA was cloned into a plasmid
vector, its identity verified by restriction endonuclease mapping and
nucleotide sequencing, and then it was used to create a size-altered
competitive template (CT) construct. The specific oligonucleotides for
the isolation and/or primary transcript assay are listed in each
section below. The construction of each CT is described below and shown
graphically in Fig. 1
.
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). The construct fidelity was confirmed by restriction
digest analysis and sequencing. Competitive template RNA was generated
by in vitro transcription (RiboMAX-T7, Promega Corp.). To ensure that RT-PCR would not amplify mRNA, a second
upstream primer was synthesized which overlapped the exon 1/intron 1
boundary (5'-AGA GAT AGC CAA CTG CAC AGG TG-3'). The downstream primer
(listed above) was used for both isolation of the FSHß gene fragment
and the FSHß PT assay. The size of the RT-PCR assay products using
these primers is 680 bp for the native RNA and 540 bp for the CT (GC
correction factor = 0.82).
2) LHß
For the LHß primary transcript CT, oligonucleotide primers
flanking intron 1 (upstream 5'-GGT ATC AAG AAT GGA GAG GCT CC-3' and
downstream 5'-GAT GCA GAC TGG GCA GAA CTC A-3') were used to amplify a
396-bp of the LHß gene containing intron 1 and 125 bp of exon 2 from
rat genomic DNA. This LHß gene fragment was inserted into the
pGEM-T-Easy expression vector (Promega Corp.). The
sequence of the LHß gene fragment was confirmed by restriction digest
analysis and sequencing. The sequence of the LHß gene fragment was
compared with the published rat LHß gene (4) for
verification. The competitive template was created by replacing a 138bp
fragment (AvrII/BsmI digest) of the LHß
intron 1 with a 260-bp sequence of pUC19 (Life Technologies, Inc., SapI/XbaI digest, Fig. 1).
The sequence of the CT was confirmed by restriction digest analysis and
sequencing. To ensure that RT-PCR would not amplify mRNA, a second
upstream primer was synthesized which overlapped the exon 1/intron 1
boundary (upstream 5'-ATG GAG AGG CTC CAG GTA AGA TG-3'). The
downstream primer used for gene isolation was also used in the PCR
assay. The size of the PCR products using these primers is 385 bp for
the native RNA and 507 bp for the CT (GC correction factor =
0.76).
3) Alpha
For the primary transcript CT, oligonucleotide primers
flanking intron 3 (up-stream primer 5'-CAC GTG CTG TGT GGC CAA ATC
AT-3', downstream primer 5'-ACT CTG GCG TTT CCC ATC ACT GT-3') were
used to amplify an approximately 1200-bp product from rat genomic DNA.
This gene fragment was inserted into to pGEM-T expression vector.
Approximately 420 bp of the 5' portion of intron 3 was sequenced
(submitted to GenBank). The sequence of 5' portion of the rat
intron 3 was 85% homologous with intron 3 of the mouse gene
(22). The CT was created by replacing a 213bp fragment
(PacI/XbaI digest) of the -intron 3 clone with
a 333bp sequence of pBR322 (Life Technologies, Inc.,
PvuI/BsaI digest, Fig. 1). The sequence of the CT
was confirmed by restriction digest analysis and sequencing. The
oligonucleotides used for the PT assay were the same upstream
primer as used to isolate the genomic DNA fragment and downstream
5'-GAG GCA GAA CCC TTT ACA G-3' (within intron 3, Fig. 1). The size of
the PCR products using these primers is 381 bp for the native RNA and
528 bp for the CT (GC correction factor = 0.61).
Conditions of gonadotropin subunit gene primary transcript RT-PCR
assays
The quantitative gonadotropin subunit primary transcript assays
are similar to our other quantitative RT-PCR assays that measure mRNA
(23, 24, 25). Each assay was tested to ensure that: 1) the
concentration of MgCl2 was optimal; 2) the
annealing and dissociation temperatures resulted in optimal DNA
amplification; and 3) that each PCR remained in the logarithmic phase
of amplification at 35 cycles (data not shown). For each RNA sample, a
PT assay includes four separate RT-PCR with a range of CT
concentrations (2, 10, 50, or 200 fg) and a constant amount of total
pituitary RNA. The reaction conditions are as follows. The native and
CT RNAs were reverse transcribed in a total volume of 20 µl
containing: 10 mM Tris-Cl, pH 8.3; 50 mM KCl; 5
mM MgCl2; deoxynucleotide
triphosphates (1 mM dATP, 1 mM cCTP, 1
mM dTTP, 1 mM dGPT); 1.75 mM random
primer (Roche Molecular Biochemicals); 5 mM
dithiothreitol; 20U RNase inhibitor (Roche Molecular Biochemicals); and 100 U Reverse Transcriptase II (Life Technologies, Inc.). The RT reaction conditions were: 5 min at
25 C for random primer annealing; 45 min at 42 C for RT; and 99 C for
10 min to stop the reaction. To each sample we added 10 µl of a
solution containing: 10 mM Tris-Cl, pH 8.3; 50
mM KCl; 5 mM MgCl2; 2
µM oligonucelotide primers (1 µM forward
primer, 1 µM reverse primer), 1 µCi
32P-dCTP; and 2.5 U Taq DNA polymerase
(Promega Corp., Madison, WI). The conditions for the PCR
were: denaturation at 94 C for 1 min followed by primer annealing and
extension at 60 C for 1 min; 35 cycles of 94 C denaturing (30 sec) and
60 C annealing and extension (1 min); finishing with 60 C for 5 min.
The PCR products were separated by electrophoresis in a 3% agarose gel
containing ethidium bromide (NuSieve 3:1 Agarose; FMC Corp., Rockland,
ME). Native and CT RNA bands were excised from the gel.
32P-dCTP incorporation was measured by
scintillation counting. The amounts of native RNA used to measure PT
concentrations using these conditions were 75, 75, and 200 ng RNA/PCR
reaction for , LHß, and FSHß subunits, respectively.
Characterization of the gonadotropin subunit gene primary
transcript RT-PCR assays
The amount of PT in each pituitary RNA was calculated as
previously described for our competitive RT-PCR mRNA assays
(23, 24, 25). Briefly, for each point in the standard curve
(i.e. CT concentration), the ratio of cpm incorporated into
the native band is compared with the total cpm incorporated (sum of the
native and competitive template bands) via the following equation:
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. The ethidium
bromide stained gel is shown on the top (A) and depict measurement of
50 ng of total pituitary RNA for an intact adult male rat
(left) and a 7-day castrated animal (right). Note
that the abundance of native product (top band) increases
whereas the abundance of CT product (bottom band) decreases
with declining starting amounts of CT RNA. The mathematical plot for
each set of quadruplicate reactions (single sample) is shown on the
bottom (B, with equations). When the equations are solved for y =
0.5, the FSH PT concentration in the intact animal was found to be 33.8
fg compared with 92.0 fg in the castrated animal.
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). Quantitative
assays for each subunit PT were performed with total RNA/PCR of 50–400
ng. With increasing amounts of RNA/PCR assay, the measured level of
gene expression was linear throughout this range.
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5 times
within a single assay to establish intraassay variability. For each of
the assays, those values are: interassay- = 22.2%, LHß
= 19.2%, FSHß = 14.1%; intraassay- = 8.9%, LHß
= 6.7%, FSHß = 5.0%.
Hormone analysis
Serum LH was measured by RIA using NIDDK LH RP-3 as standard.
The range of this assay is 0.0625–32 ng/ml and the intra and
interassay variabilities are 10.9% and 16.1% respectively
(26).
Statistics
The results of all experiments were analyzed by ANOVA.
Differences between treatments were determined by Dunnett’s criterion
(27) with either cast + T or intact male rats as the
controls. The determination of the half-lives of LHß and FSHß PT
following administration of GnRH antagonist to 7-day castrate males was
based on a negative exponential decay model in which the parameters
were estimated by nonlinear regression. Before analyses, all
measurements were transformed to the natural logarithmic scale to
attain equal the residual variation among treatments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
LHß, and FSHß mRNA and PT concentrations were determined. Results
are shown in Fig. 4.
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> FSHß LHß. Expectedly, expression of each mRNA
species was 50- to 120-fold higher than the corresponding PT species
(: 
100-fold, LHß: 50-fold, FSHß: 120-fold). Following
castration, LH secretion increased 15- to 20-fold. Significant
increases in -mRNA (3-fold), LHß mRNA (6-fold) and FSHß mRNA
(3-fold) were observed, similar to previous reports (19, 28, 29). Following castration, all three PT concentrations
increased, though the 60% rise in did not reach statistical
significance. In contrast, significant increases in LHß PT (6-fold)
and FSHß PT (3-fold) were observed.
Exp 2: the determination of the half-disappearance rate of
gonadotropin subunit PTs in castrate male rats following GnRH
blockade.
To determine the half-disappearance rate of subunit PTs, 7-day
castrate animals were treated with the GnRH antagonist LRF-147 and
killed 0.5, 2, 8, or 16 h later. mRNA concentrations did not
change over this time period (data not shown). PT concentrations
did not change significantly after GnRH antagonist (Fig. 5). In contrast, LHß PT concentration
fell rapidly after GnRH blockade with a half-disappearance time
of approximately 2.7 h. FSHß PT concentrations fell more
rapidly, having a half-disappearance time of 0.8 h. Within 16
h, FSHß and LHß PT concentrations had returned to levels similar to
intact animals.
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, LHß, and
FSHß primary transcript (PT) concentrations were measured in each
animal (Fig. 6). Gonadotropin subunit
mRNA concentrations were not measured; as previous data suggests that
this time course is insufficient to observe GnRH-stimulated responses
(14).
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PT > FSHß LHß (compare Figs. 4 and 6). Although PT levels increased approximately 60% within 5 min
after a GnRH pulse, this increase did not reach statistical
significance. Levels of PT tended to decline thereafter through 30
min. In contrast, exogenous GnRH markedly increased both LHß and
FSHß PT levels. LHß PT was 6-fold higher within 5 min then declined
through 30 min. FSHß PT rose approximately 4-fold within 5 min then
declined more rapidly than LHß, decreasing approximately 60% by 30
min though values remained above those in control animals. The more
rapid decline in FSHß PT is consistent with the data presented in
Fig. 5, in which FSHß PT was cleared more rapidly than
LHß PT after treatment with a GnRH antagonist. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), as measured by
the concentration of primary transcript RNAs, were increased after
castration and paralleled changes in mRNA concentration
(11). Blockade of GnRH rapidly reduced PT concentrations,
before changes in mRNA levels, with half-disappearance times for LHß
PT and FSHß PT of 2.7 h and 0.8 h, respectively. Moreover,
blockade of GnRH action restored PT concentrations to the range of
values seen in intact animals within 16 h. Finally, exogenous GnRH
pulses rapidly (within 5 min) increased LHß and FSHß PT
concentrations in a GnRH-deficient animal model. Thus, mRNA synthesis
is GnRH-dependent as has been reported for steady-state mRNA
concentrations.
To date, few analyses of hormonal regulation of gonadotropin subunit
gene transcription rates have been reported (11, 14, 15),
in large part reflecting the limitations of nuclear run-on assays.
Nuclear run-on assays are costly; requiring pituitaries from three to
four animals to provide sufficient nuclear material for a single
measurement of transcription rates, and do not allow for the
simultaneous measurement of mature mRNA. In addition, quantification of
incorporated 32P-UTP in the run-on assays used a
hybridization step in which RNA became bound to cDNA fixed on a
nitrocellulose filter, allowing only pg sensitivity. Also of import to
physiologic studies, samples could not be repeatedly measured in
separate assays, thereby prohibiting assessment of interassay
variability and requiring that all samples be measured together. To
address these issues, we developed highly sensitive and reproducible
quantitative RT-PCR assays for each of the gonadotropin subunit PTs,
having similar inter and intraassay variability to our previously
reported RT-PCR assays for follistatin, inhibin/activin subunit, and
activin receptor subunits (23, 24, 25). Using these systems,
we are able to measure all three gonadotropin subunit mRNA and PT RNA
concentrations within the RNA derived from a single pituitary gland.
Routinely, we recover 70–90 µg of total RNA/pituitary in male and
female rats. The dot-blot hybridizations require 25 µg for duplicate
measurements of , LHß, and FSHß mRNA, and the PT assays 2.5 µg
for measurement of , LHß, and FSHß PTs. Thus, RNA recovery is
sufficient to allow replicate measurements with residual RNA available
to expand investigation of gonadotrope physiology should other genes of
interest be identified.
The oligonucleotide primers for each assay were selected to direct PCR amplification such that the amplified region would encompass both exon and intron sequences. This strategy determined that amplification of mRNA-derived cDNAs would not be favored and thereby not interfere with PT quantification. Conversely, the concentration of PT RNAs in the total RNA extracted is low and provides a minimal (<10%) contribution to the dot-blot hybridizations used for mRNA quantification. Thus, these two methodologies allow independent assessment of sequential changes in gene expression. Indeed, changes in primary transcript concentration precede alterations in mRNA concentration both after exogenous GnRH pulses (current data and Ref. 14) and after treatment with a GnRH antagonist in castrated rats, suggesting that alterations in gene transcription (mRNA synthesis) determine, at least in large part, steady-state mRNA concentrations.
Both orchidectomy and ovariectomy, have been shown to increase
gonadotropin subunit gene transcription (11, 30, 31).
Specifically, in 5-day castrate male rats, increased transcription of
(3-fold), LHß (5-fold), and FSHß (5-fold) were noted. The
current data reveal a similar trend for PT and identify increases
of similar magnitude for the ß subunit PTs. Using a similar protocol
of iv GnRH antagonist injections, we previously showed that the
half-disappearance time for the gonadotropin subunit mRNAs was
50 h, LHß = 65 h, FSHß = 20 h,
(11). Consistent with these findings, the
half-disappearance time of the FSHß PT is shorter than LH ß PT (0.8
vs. 2.7 h, respectively), and additionally the decline
in PT levels precedes the fall in mRNA concentrations.
We previously used run-on assays to document an increase in mRNA
synthesis 20 min after an exogenous GnRH pulse (14). The
present results expand on those findings with a sequential assessment
of gonadotrope responses during the first 30 min following a GnRH
pulse. The run-on assay revealed approximately 3-fold increases for
each gonadotropin subunit transcription rate (14). Using
quantitative RT-PCR, we observed a response of similar magnitude within
5 min for FSH ß PT and a larger (6-fold) response for LH ß PT. As
described following castration, PT levels tended to increase after
pulsatile GnRH though changes did not reach statistical significance.
The explanation for this modest difference in transcription between
the current and prior studies remains uncertain but may reflect the
inherent difficulty in measuring gene expression (regardless of the
methodology) in light of its presence in both gonadotropes and
thyrotropes. Also as changes in PT after castration are modest,
this suggests that gene expression in thyrotropes (not regulated by
GnRH) is a significant proportion of the basal values observed.
Following a GnRH pulse, both ß subunit PT concentrations were
increased maximally within 5 min, indicating that postreceptor
signaling from cell membrane to the nucleus by GnRH is rapid and occurs
earlier than the 20-min interval previously reported (14).
After the initial increase at 5 min, the decline in FSHß PT was more
rapid than for LHß, in agreement with results in castrate animals
following GnRH blockade. These findings extend and refine prior data
(14) and suggest that transcriptional responses to each
GnRH pulse is of brief duration, likely being completed in
approximately 1 h or less. Of interest, we have previously
reported that the response in ß subunit transcription appears to
diminish over time, being absent after 24 h of GnRH pulses despite
increasing mRNA concentrations (14). Those data suggested
that, with longer treatment regimens, transcription may occur in bursts
(i.e. be intermittent), and it is uncertain whether each
GnRH pulse induces a transcriptional response. Currently, studies are
underway in our laboratory to explore responses to an ongoing GnRH
stimulus and determine whether longer exposures to GnRH are associated
with consistent transcriptional responses and/or are needed to increase
PT expression.
In summary, the use of quantitative RT-PCR assays for measurement of primary transcript RNAs allows assessment of both transcription rates and mRNA concentrations for all three gonadotropin subunits within RNA from a single rat pituitary. This technique will enhance our ability to study gonadotrope physiology and thereby detail the sequence of cellular events following hormonal signals. The present results show that GnRH-dependent changes in gene transcription are rapid and precede alterations in mRNA concentration, providing further support for the notion that transcriptional regulation is a major determinant of mRNA expression.
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Footnotes |
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Received May 18, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit of
rat pituitary glycoprotein hormone. Primary structure of the precursor
determined from the nucleotide sequence of cloned cDNAs. J Biol
Chem 257:8368–8371 and LH ß-mRNA in male and
female rats after castration: quantitation using an optimized RNA
dot-blot hybridization assay. Endocrinology 119:691–698[Abstract]
-subunit gene of the pituitary
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H. Zapatero-Caballero, F. Sanchez-Franco, C. Fernandez-Mendez, M. Garcia-San Frutos, L. M. Botella-Cubells, and G. Fernandez-Vazquez Gonadotropin-Releasing Hormone Receptor Gene Expression During Pubertal Development of Female Rats Biol Reprod, February 1, 2004; 70(2): 348 - 355. [Abstract] [Full Text] [PDF]
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 D. Coss, S. B. R. Jacobs, C. E. Bender, and P. L. Mellon A Novel AP-1 Site Is Critical for Maximal Induction of the Follicle-stimulating Hormone {beta} Gene by Gonadotropin-releasing Hormone J. Biol. Chem., January 2, 2004; 279(1): 152 - 162. [Abstract] [Full Text] [PDF]
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L. L. Burger, D. J. Haisenleder, K. W. Aylor, A. C. Dalkin, K. A Prendergast, and J. C. Marshall Regulation of Luteinizing Hormone-{beta} and Follicle-Stimulating Hormone (FSH)-{beta} Gene Transcription by Androgens: Testosterone Directly Stimulates FSH-{beta} Transcription Independent from Its Role on Follistatin Gene Expression Endocrinology, January 1, 2004; 145(1): 71 - 78. [Abstract] [Full Text] [PDF]
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D. J. Haisenleder, L. L. Burger, K. W. Aylor, A. C. Dalkin, and J. C. Marshall Gonadotropin-Releasing Hormone Stimulation of Gonadotropin Subunit Transcription: Evidence for the Involvement of Calcium/Calmodulin-Dependent Kinase II (Ca/CAMK II) Activation in Rat Pituitaries Endocrinology, July 1, 2003; 144(7): 2768 - 2774. [Abstract] [Full Text] [PDF]
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H. Zapatero-Caballero, F. Sanchez-Franco, N. Guerra-Perez, C. Fernandez-Mendez, and G. Fernandez-Vazquez Gonadotropin-Releasing Hormone Receptor Gene Expression During Pubertal Development of Male Rats Biol Reprod, May 1, 2003; 68(5): 1764 - 1770. [Abstract] [Full Text] [PDF]
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L. L. Burger, A. C. Dalkin, K. W. Aylor, D. J. Haisenleder, and J. C. Marshall GnRH Pulse Frequency Modulation of Gonadotropin Subunit Gene Transcription in Normal Gonadotropes--Assessment by Primary Transcript Assay Provides Evidence for Roles of GnRH and Follistatin Endocrinology, September 1, 2002; 143(9): 3243 - 3249. [Abstract] [Full Text] [PDF]
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B. A. Adams, E. D. Vickers, C. Warby, M. Park, W. H. Fischer, A. Grey Craig, J. E. Rivier, and N. M. Sherwood Three Forms of Gonadotropin-Releasing Hormone, Including a Novel Form, in a Basal Salmonid, Coregonus clupeaformis Biol Reprod, July 1, 2002; 67(1): 232 - 239. [Abstract] [Full Text] [PDF]
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F.E.M. Rebers, G.A.M. Hassing, W. van Dijk, E. van Straaten, H.J.Th. Goos, and R.W. Schulz Gonadotropin-Releasing Hormone Does Not Directly Stimulate Luteinizing Hormone Biosynthesis in Male African Catfish Biol Reprod, June 1, 2002; 66(6): 1604 - 1611. [Abstract] [Full Text] [PDF]
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D.J. Haisenleder, L.J. Workman, L.L. Burger, K.W. Aylor, A.C. Dalkin, and J.C. Marshall Gonadotropin Subunit Transcriptional Responses to Calcium Signals in the Rat: Evidence for Regulation by Pulse Frequency Biol Reprod, December 1, 2001; 65(6): 1789 - 1793. [Abstract] [Full Text] [PDF]
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L. L. Burger, A. C. Dalkin, K. W. Aylor, L. J. Workman, D. J. Haisenleder, and J. C. Marshall Regulation of Gonadotropin Subunit Transcription after Ovariectomy in the Rat: Measurement of Subunit Primary Transcripts Reveals Differential Roles of GnRH and Inhibin Endocrinology, August 1, 2001; 142(8): 3435 - 3442. [Abstract] [Full Text] [PDF]
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