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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: Laura L. Burger, University of Virginia, Department of Internal Medicine, P.O. Box 801387, Charlottesville, Virginia 22908. E-mail: llb3k{at}virginia.edu
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Abstract |
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Following ovariectomy, LHß PT concentrations increased 2- to
3-fold between 72 h and 7 d, paralleling changes in serum LH
and LHß mRNA. In contrast, serum FSH, FSHß mRNA, and FSHß
PT concentrations were 6- to 9-fold greater 12–24 h after
ovariectomy followed by an additional 2.5-fold increase at 72 h.
Although 
RNA was elevated at 72 h after ovariectomy,
-primary transcript did not change. GnRH antagonist prevented the
increase in LHß-PT at 72 h, but had no effect on the increase in
FSHßPT at 12 h and was only partially effective at 72 h.
The acute GnRH-independent increase in FSHß-primary transcript after
ovariectomy could be duplicated by the administration of inhibin
antiserum to intact rats; inhibin- antiserum did not affect
LHß-primary transcript, but increased FSHß-primary transcript
concentrations 8- to 11-fold.
The half-disappearance rates of LHß and FSHß primary transcripts were measured after GnRH blockade or administration of recombinant human inhibin A. The half-disappearance times for LHß and FSHß primary transcripts following GnRH blockade were 13 and 17 min, respectively; the mRNAs did not change. The effects of inhibin were specific for FSHß; 60 min after inhibin FSHß-primary transcript was undetectable with a half-disappearance time of 19 min, additionally FSHß mRNA levels also fell with a half-life of 94 min.
In conclusion, these data support previous evidence that GnRH regulates
gonadotropin gene expression primarily at the level of transcription.
However, the acute increase in FSHß-primary transcript after
ovariectomy or immunoneutralization of inhibin-, and the rapid fall
in FSHß-primary transcript following rh inhibin, provide novel
evidence that inhibin suppresses FSHß gene transcription in addition
to its action in regulating FSHß mRNA stability.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and unique ß-subunits, the latter conferring
biological activity. In the rat, distinct genes located on different
chromosomes code for the three gonadotropin subunits (1).
Pulsatile release of the decapeptide GnRH is the primary regulator of
the three gonadotropin subunit mRNAs. GnRH differentially regulates
gonadotropin subunit mRNA expression through alterations in both pulse
amplitude and frequency (2, 3). Additionally in female
rats, the peptide hormones inhibin, activin, and follistatin also
regulate FSHß mRNA expression (3, 4, 5, 6, 7). Inhibin and activin are members of the TGFß family and have antagonistic functions. Inhibin is produced primarily in the gonads (4) and acts on the pituitary to suppress FSHß mRNA expression (5, 6, 7). Activin is produced by both the gonads and gonadotropes (4) and works through specific activin receptors (8) to increase FSHß mRNA expression (9), in part via gene transcription (10). Follistatin, an unrelated glycopeptide hormone, is produced by the gonads, gonadotropes, and folliculo-stellate cells (11) and decreases FSHß mRNA expression by binding to and bioneutralizing activin (12, 13).
Gonadotropin subunit gene expression is differentially regulated
following ovariectomy (OVX). Serum LH rises slowly after OVX
(reflecting an increase in GnRH secretion) and is followed by increases
in and LHß mRNA expression around 3–4 d post OVX, which can be
prevented by treatment with a GnRH antagonist (6, 14, 15, 16).
In contrast, serum FSH and FSHß mRNA rise rapidly after OVX. FSHß
mRNA doubles within 1 h of OVX and serum FSH is elevated by 8
h, even in the presence of a GnRH antagonist (6, 16, 17).
Similar results are found when inhibin- antisera is administered to
intact female rats, demonstrating that the GnRH-independent increases
in FSH and FSHß mRNA that occur immediately after OVX result from the
loss of circulating inhibin (6, 7). Additionally, there is
a second increase in FSH and FSHß mRNA, coincident with the increases
in and LHß mRNA, which is abolished by GnRH antagonist (6, 14, 15, 16). These selective changes in gonadotropin subunit mRNA
concentrations may reflect alterations in transcription and/or mRNA
stability.
GnRH appears to exert its main action on the gonadotropin subunit genes
by regulating mRNA synthesis (transcription). We have shown that GnRH
pulses increase subunit transcription in a GnRH-deficient male rat
model, although the transcriptional responses were not maintained
(18). Subunit gene transcription rates are elevated in
long term (>14 d) OVX rats (19, 20, 21, 22), and the
post OVX increases in gonadotropin subunit gene transcription are GnRH
dependent. Administration of a GnRH antagonist reduced and LHß
significantly but had only modest effects on FSHß (21, 22), hinting at roles for activin, inhibin, and or/follistatin
in the regulation of FSHß gene transcription. The mechanism(s)
whereby inhibin, activin, and follistatin regulate FSHß mRNA
expression remain uncertain and may involve changes in both
transcription and/or mRNA stability (6, 7, 9, 10, 23, 24, 25).
Previously, gonadotropin subunit gene transcription was measured using nuclear run-on assays, which have several limitations. These include the need to use nuclei from three to four pituitaries for a single measurement, precluding repeated measurements, and mRNA concentrations cannot be determined as it is difficult to isolate both nuclei and cytoplasmic RNA simultaneously. We have recently developed quantitative RT-PCR assays to measure subunit primary transcript (PT) RNA (26). The assays are based on oligonucleotide primers flanking intron-exon boundaries and require only nanogram amounts of RNA, allowing us to measure PT and steady-state mRNA levels in a single pituitary RNA sample. We have shown that changes in subunit PTs precede changes in mRNA and PT half-disappearance times are short and therefore provide an index of transcription. Importantly, subunit PT concentrations change in a physiologically appropriate manner, and LHß and FSHß PT concentrations were 6- and 3-fold greater in male rats 7 d after castration and rapidly fell to basal levels after a GnRH antagonist. Additionally, in a GnRH-deficient male rat model, LHß and FSHß PT increased 4- to 6-fold 5 min after a GnRH pulse before declining to levels seen in control animals.
There are two aims in this study. First, we investigated the changes in gonadotropin subunit gene transcription in the 7 d after OVX during the dynamic period following the loss of gonadal feedback. Second, we aimed to determine the relative contributions of inhibin and GnRH on subunit gene transcription following OVX.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Exp 1: measurement of gonadotropin subunit primary transcript
concentrations after OVX
Groups of intact female rats and rats ovariectomized for 12
h, 24 h, 72 h, and 7 d (n = 5–9/group) were
killed, and serum LH and FSH, subunit mRNA, and subunit PT
concentrations were determined.
Exp 2: to define the relative contributions of GnRH in the postOVX
increase in gonadotropin subunit primary transcripts
mRNA and PT concentrations were determined in pituitary RNA from
intact female rats and from 12 h or 72 h OVX rats ±
treatment with the GnRH antagonist LRF-147 (n = 5–7/group). At
the time of OVX, the 12 h groups were fitted with indwelling
jugular cannulas. Immediately following recovery from anesthesia, and
again 8 h later, rats received either 30 µg of the water soluble
GnRH antagonist LRF-147, iv (in 0.5 ml 0.9% saline-0.1% BSA; kindly
provided by Dr. Jean Rivier, The Salk Institute, La Jolla,
CA) or saline-BSA vehicle. Administration of 30 µg LRF-147 iv every
8 h to 14 d castrate male rats reduced serum LH to basal by
2 h and maintained suppression for 8–12 h (26, 27).
The 72 h OVX groups were treated with either 100 µg of LRF-147
sc (in 0.5 ml 0.9% saline-0.1% BSA) or saline-BSA vehicle every
12 h. This dose of LRF-147 sc reduces serum LH in 7 d
castrate male rats to basal levels by 24 h (our unpublished
observation). At the times indicated, rats were killed, and serum LH
and FSH, subunit mRNA, and subunit PT concentrations were
determined.
Exp 3: to assess the regulation of FSHß primary transcript by
inhibin
To determined if the loss of inhibin after OVX alters subunit PT
concentrations, intact female rats were treated for 2 or 12 h with
either sheep antiserum directed against rat inhibin -subunit (kindly
provided by Dr. W. Vale, The Salk Institute, La Jolla, CA)
or normal sheep sera (NSS, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; n = 4–7/group). Administration of
this inhibin- antiserum to intact female rats has been reported to
increase serum FSH and pituitary FSHß mRNA by 12 and 2 h,
respectively (6, 7). The day before treatment, rats
assigned to the inhibin antiserum, and NSS treatment groups were fitted
with indwelling jugular cannulas. Rats received a single injection of
either 0.5 ml inhibin antiserum or NSS and were killed 2 h or
12 h later. Serum LH and FSH, ß-subunit mRNA concentrations, and
ß-subunit PT concentrations were determined. Alpha mRNA and PT were
not measured in this experiment because Exp 1 and 2 demonstrated that
the acute loss of inhibin following OVX did not affect mRNA or PT
concentrations.
Exp 4: to determine the half-disappearance times of LHß and
FSHß primary transcripts in OVX female rats after GnRH blockade
Recently we reported that the half-disappearance times for LHß
and FSHß PT in 7-d castrate male rats following the GnRH antagonist
LRF-147 (30 µg, iv) were 2.7 h (162 min) and 0.75 h (45
min), respectively (26). To determine if there are
differences between sexes in gonadotropin PT clearance, LHß and
FSHß PT concentrations were measured in 7 d OVX rats following GnRH
blockade. Female rats were ovariectomized, and 6 d later right
jugular vein catheters were surgically inserted. On d 7, a 200 µl
blood sample was collected and a single dose of LRF-147 (30 µg/0.5 ml
0.9% saline-0.1% BSA) was administered iv. Rats were killed 0, 30,
60, and 120 min later (n = 5–6/time). A group of intact controls
was also included (n = 6). Serum LH and FSH, ß-subunit mRNA, and
ß-subunit PT concentrations were measured. -PT was not measured;
as -PT concentration was unchanged in either sex in after
gonadectomy (26 , see results for Exp 1).
Exp 5: to determine the half-disappearance times of LHß and
FSHß primary transcripts in OVX female rats after administration of
inhibin
Inhibin has been reported to decrease FSHß mRNA concentrations
in vitro both at the level of transcription and/or through
posttranscriptional mechanisms (23, 24, 25). To determine if
inhibin directly affects FSHß transcription, FSHß PT (and LHß PT
as a control) disappearance was measured following recombinant human
inhibin A (subsequently referred to as rh inhibin) to 12 h OVX
rats. Female rats were ovariectomized, and a right jugular cannula was
inserted. To eliminate endogenous GnRH action, the GnRH antagonist
LRF-147 (100 µg/0.5 ml 0.9% saline-0.1% BSA, sc) was given after
surgery and again 11 h later. At 12 h post OVX, a 200 µl
blood sample was collected and a single dose of rh inhibin (10 µg/0.5
ml 0.9% saline, iv, kindly provided by Dr. De Kretser, Monash Medical
Center, Melbourne, Australia in cooperation with Biotech Australia Pty.
Ltd. 28) was given, and rats were killed 0, 15, 30, 60,
120, and 240 min later (n = 5–8/time). DePaolo and co-workers
(29) reported that 10 µg inhibin reduced serum FSH 40%
at 4 h in 7 d OVX rats. Intact controls were also included in
this experiment (n = 5). Serum LH and FSH, ß-subunit mRNA, and
ß-subunit PT concentrations were measured.
Measurement of serum gonadotropins, RNA preparation, measurement of
subunit mRNAs, and subunit primary transcripts
Serum LH and FSH were measured by RIA using reagents provided by
the National Hormone and Pituitary Program. The RIA standards were
NIDDK RP-3 for LH and NIDDK RP-2 for FSH. The sensitivities for the LH
and FSH assays are 0.09 ng/ml and 0.8 ng/ml, respectively. The
coefficients of variation for the LH assay are 10.9% and 16.1% (intra
and interassay), and 5.3% and 12.4% for the FSH assay. Total
pituitary RNA was extracted using the acid guanidinium method
(30). 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 PCR in the absence of a
preceding reverse transcription reaction. This RNA preparation method
does not allow for the quantitation of pituitary DNA; therefore we have
expressed mRNA and PT concentrations as fmol/100 µg pituitary RNA.
Subunit mRNA concentrations were determined by dot blot hybridization
assays using 3–4 µg pituitary RNA per dot (14, 31), and
a sense strand RNA standard curve spotted on each nitrocellulose filter
(26). Subunit PT concentrations were determined by
quantitative RT-PCR assays previously described (26).
Briefly, for each subunit, regions of intron/exon were amplified
using specific oligonucleotide primers, and a size altered competitive
template RNA (CT) was made for each gene. A four point standard curve
was generated by adding a fixed amount of pituitary RNA (50–400
ng/reaction) to a graduated amount (2, 10, 50, 250 fg) of CT. The
pituitary and CT RNA were reverse transcribed followed by 35 cycles of
PCR in the presence of 32P-dCTP. The PCR products
were separated by electrophoresis in 3% agarose, the bands excised,
and 32P-dCTP incorporation determined by
scintillation counting.
Statistics
The results of the three experiments were analyzed by
multivariate and univariate ANOVA. For each experiment, a MANOVA
(32) was conducted to simultaneously test if any of the
univariate responses were influenced by treatment. Contingent on the
MANOVA findings, univariate analyses were performed by either one-way
or two-way ANOVA. Differences between treatments were determined by
Tukey’s honest significance difference criterion (33)
with an experimental type 1 error rate of 0.05. The determination of
the half-lives of FSHß mRNA, LHß PT, or FSHß PT 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 variation
among treatments. Statistical computations were carried out in SAS 6.12
(SAS/STAT Software Changes And Enhancements, 1997).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Consistent with our earlier
observations, serum LH, LHß mRNA, and mRNA expression were
unchanged until 72 h after OVX, then rose through d 7. In
contrast, serum FSH and FSHß mRNA were elevated by 12 h,
followed by a second increase at 72 h. Changes in LHß and FSHß
PT concentrations parallel the changes in serum gonadotropins and
ß-subunit mRNAs after OVX. LHß PT was 3.5-fold greater than in
intact rats 72 h after OVX and remained elevated through d 7.
FSHß PT was 6- to 9-fold greater 12–24 h after OVX, followed by an
additional 2.5-fold increase at 72 h. Although mRNA was
increased 72 h after OVX, -PT did not change.
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Consistent with our earlier findings, serum LH, LHß mRNA, and
mRNA were elevated at 72 h and were abolished by GnRH antagonist.
Serum FSH and FSHß mRNA increased in a biphasic manner post OVX, with
increases at both 12 and 72 h. Antagonist partially prevented the
rise in serum FSH at 12 h OVX but did not affect the increase in
FSHß mRNA at 12 h and only partially prevented the rise at
72 h. The changes in LHß and FSHß PTs paralleled those in
serum gonadotropins and ß-subunit mRNAs. LHß PT concentration was
increased at 72 h and was abolished by antagonist. FSHß PT was
increased at 12 h, even in the presence of antagonist, and rose a
second time at 72 h. The 72 h increase in FSHß PT was
significantly attenuated by antagonist but remained 10-fold greater
than intact animals. As in Exp 1, -PT did change after OVX, and
levels were unaltered by GnRH antagonist.
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antiserum on serum gonadotropins and
ß-subunit mRNAs and PT are presented in Fig. 3. The actions of the inhibin-
antiserum were specific for FSH and FSHß mRNA, which were increased
at 12 and 2 h respectively. Inhibin antiserum did not increase
LHß PT, but NSS suppressed LHß PT (and LH at 12 h) compared
with antiserum treated groups. The mechanisms of this action are
unknown, and may reflect compounds in the NSS that suppress endogenous
GnRH. As with FSH and FSHß mRNA, inhibin- antiserum increased
FSHß PT 8- to 11-fold at 2 and 12 h.
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. LRF-147 reduced serum LH by 30 min,
and levels were the same as controls at 120 min. FSH tended to decrease
after antagonist but was only significantly lower at 60 min, reflective
of the longer half-life of circulating FSH. Antagonist had no affect on
either LHß or FSHß mRNA concentrations by 120 min. In contrast,
both LHß and FSHß PT fell rapidly after GnRH blockade with
half-disappearance times of 13 and 17 min, respectively. PT
concentrations did not fall to intact control values by 120 min, which
is consistent with data we reported for male rats
(26).
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. GnRH
antagonist reduced serum LH concentrations to values at assay
sensitivity. FSH increased 3-fold 12 h after OVX even with GnRH
blockade. Inhibin did not reduce serum FSH, which may reflect the short
duration of this experiment; previous studies found that the actions of
inhibin on FSH were delayed and FSH was not reduced before 4 h
(29, 34, 35). GnRH antagonist reduced 12 h OVX LHß
mRNA and PT concentrations to intact or lower values, and rh inhibin
had no additional effects. In contrast, both FSHß mRNA and PT
concentrations were 2-fold greater than intact rats at 12 h OVX
even in the presence of a GnRH antagonist. Both FSHß mRNA and PT fell
rapidly after rh inhibin with half-disappearance times of 94 and 19
min, respectively. The effects of inhibin were dramatic; FSHß mRNA
and PT were reduced to intact levels or below by 120 and 30 min,
respectively, and FSHß PT was undetectable by 60 min.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-subunit transcription
(22, 18). Pulsatile signals are essential; and GnRH
differentially regulates subunit gene transcription via changes in
pulse frequency. Fast physiological pulse frequencies (8- to 30-min
pulse intervals) favor stimulation of transcription, intermediate
pulse frequencies (every 30 min) maximally stimulate LHß
transcription, and slower frequencies (every 120 min) only stimulate
FSHß transcription (18).
GnRH also regulates subunit gene transcription rates after gonadectomy.
In male rats, LHß and FSHß transcription increased 4- to 6-fold
7 d after castration (26, 27) and could be prevented
by GnRH antagonist (27). The effects of castration on
transcription were variable; either increasing (27) or
remaining unchanged (26). In female rats, Shupnik and
co-workers (19, 21) reported that , LHß, and FSHß
gene transcription rates, measured by nuclear-run assays, increased
2.5-, 10-, and 3.5-fold, respectively, in long term (28–40 d) OVX
rats. GnRH blockade prevented the increases in and LHß
transcription but only partially prevented the rise in FSHß
(21). Our data using RT-PCR assays to measure PT
concentrations support these findings. After OVX, LHß PT increased
3.5-fold at 72 h; and FSHß PT rose 6- to 9-fold between 12–24 h
and increased an additional 2.5-fold at 72 h. GnRH antagonist
prevented the increase in LHß PT but had no effect on the increase in
FSHß PT at 12 h and only partially reduced the later increase at
72 h. In contrast, we did not see any consistent changes in -PT
after OVX, which may reflect that both gonadotropes and thyrotropes
produce -subunit and thus regulation of gene expression is only
partially GnRH dependent.
The finding of increased FSHß PT at 12 h after OVX or 2 h following inhibin immunoneutralization contrasts prior data obtained with run-on assays, where FSHß transcription rates were not increased 2–12 h after OVX (6). However, FSHß mRNA rose 2- to 3-fold after OVX or inhibin neutralization, and we previously concluded that the major role of inhibin was in regulating the rate of FSHß mRNA disappearance. There are a number of potential explanations for these contrasting results. The PT assays may be more sensitive in detecting changes in gene transcription. The magnitude of change in FSHß PT increases with time after OVX, and as RT-PCR measures PT at fmol concentrations it may detect alterations earlier than run-on assays, which rely on binding kinetics similar to dot-blot hybridizations for measuring mRNA. Also, changes in both transcription rate and mRNA half-life may occur after a decline in circulating inhibin. To address this possibility, we determined the disappearance of FSHß PT in vivo by two methods, following GnRH blockade and inhibin administration.
The half-disappearance times for LHß and FSHß PT following GnRH blockade in OVX female rats were 13 and 17 min respectively. For LHß PT the half-life in OVX females is significantly shorter than the 162 min (2.7 h) we reported in castrate males (26). It is unclear if the difference between the sexes is due to differences in: experimental duration (120 min vs. 16 h with few early measurements), previous steroid exposure, and/or male and female gonadotropes. The 17-min half-life of FSHß PT in females is probably similar to the 45 min time in castrate males. LHß and FSHß mRNAs were unchanged after GnRH antagonist, which reflects the long half-lives of LHß and FSHß mRNAs previously reported as 65 and 20 h, respectively (27). The effects of rh inhibin were specific to the FSHß subunit; FSHß mRNA and PT fell rapidly with half-disappearance times of 94 and 19 min respectively. The half-lives for FSHß PT were identical after either GnRH blockade or rh inhibin. As GnRH antagonists inhibit FSHß transcription, these data support the concept that inhibin also regulates FSHß transcription. However, the 94-min half-life of FSHß mRNA after rh inhibin is significantly shorter than the 20 h half-life reported after GnRH blockade (27), indicating that inhibin also decreases FSHß mRNA stability.
The effects of rh inhibin on both FSHß transcription and mRNA stability are consistent with earlier reports. In pituitary cells from transgenic mice containing the promoter plus the first intron of the ovine FSHß gene inhibin reduced FSHß promoter activity by 80–90% (36). In vivo, Clarke et al. (23) reported that bovine inhibin given to sheep reduced FSHß transcription rates by 50% and also reduced FSHß mRNA by 100%, suggesting actions at both transcriptional and posttranscriptional levels. Also, Attardi et al. (24) reported that the half-disappearance of FSHß mRNA following blockade of transcription in vitro was not altered by the addition of inhibin, suggesting that inhibin acts at the level of transcription. In addition, the suppressive effects of inhibin on FSHß mRNA expression could be blunted by inhibitors of translation; and the authors proposed that inhibin may induce the transcription of a protein that reduces the stability of FSHß mRNA (25). While little is known regarding the degradation and processing rate of the FSHß PT, it is also possible that in addition to regulating the FSHß mRNA half-life inhibin may also regulate the half-life of FSHß PT.
The mechanism(s) used by inhibin to regulate FSHß gene transcription are unknown. Inhibin and activin have reciprocal actions on FSHß mRNA expression, and a recent report showed that activin increases FSHß transcription in vitro, as measured by changes in FSHß PT (10). Potentially, inhibin could regulate production of pituitary-derived activin, but two findings make this unlikely. The modest increase in pituitary activin ßB mRNA 12 h after OVX can be prevented by a GnRH antagonist and ßB mRNA levels do not change after administration of inhibin antiserum (7). These data, combined with recent reports of putative inhibin receptors (37, 38, 39, 40), make it probable that inhibin acts through its own membrane receptor, either directly or by impairing the actions of activin, to modify FSHß gene transcription and mRNA stability.
In conclusion, the current data show that LHß and FSHß transcription are differentially regulated following OVX. The GnRH-dependent increases in LHß and FSHß PT at 72 h after OVX provide further evidence that GnRH regulates gonadotropin gene expression at the level of transcription. Our findings also provide novel evidence that inhibin suppresses FSHß transcription as indicated by: 1) the acute GnRH independent increase in FSHß PT that occur immediately after OVX; 2) the increase in FSHß PT after inhibin immunoneutralization; and 3) the rapid and profound suppression of FSHß PT by rh inhibin in OVX rats. Additionally, the rapid disappearance of FSHß mRNA after rh inhibin provides further evidence that inhibin not only regulates FSHß transcription but also mRNA stability.
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Acknowledgments |
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Footnotes |
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Abbreviations: CT, Competitive template; NSS, normal sheep sera; OVX, ovariectomy; PT, primary transcript.
Received February 9, 2001.
Accepted for publication April 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, ß A, and ß B subunits
in various tissues predicts diverse functions. Proc Natl Acad Sci USA 85:247–251 and luteinizing hormone ß
messenger ribonucleic acid (RNA) of male and female rats after
castration: quantitation using an optimized RNA dot blot hybridization
assay. Endocrinology 119:691–698-subunit knockout mice. J Biol Chem 273:398–403This article has been cited by other articles:
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 C. Welt, Y. Sidis, H. Keutmann, and A. Schneyer Activins, Inhibins, and Follistatins: From Endocrinology to Signaling. A Paradigm for the New Millennium Experimental Biology and Medicine, October 1, 2002; 227(9): 724 - 752. [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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