The Frequency of GonadotropinReleasing Hormone Secretion
Regulates Expression of a and
Luteinizing Hormone /?-Subunit
Messenger Ribonucleic Acids in
Male Rats
D. J. Haisenleder, S. Khoury, S. M. Zmeili, S. Papavasiliou,
G. A. Ortolano, C. Dee, J. A. Duncan, and J. C. Marshall
Division of Endocrinology and Metabolism
Department of Internal Medicine
University of Michigan Medical Center
Ann Arbor, Michigan 48109
The influence of GnRH pulse frequency on LH subunit mRNA concentrations was examined in castrate,
testosterone-replaced male rats. GnRH pulses (25
ng/pulse) or saline to controls, were given via a
carotid cannula at intervals of 7.5-240 min for 48 h.
a and LH/3 mRNA concentrations were 109 ± 23 and
30 ± 5 pg cDNA bound/100 M9 pituitary DNA, respectively, in saline controls. GnRH pulse intervals
of 15,30, and 60 min resulted in elevated a and LH/?
mRNAs (P < 0.01) and maximum responses (4-fold,
a; 3-fold, LH/3) were seen after the 30-min pulses.
Acute LH release to the last GnRH pulse was seen
after the 15-, 30-, and 60-min pulse intervals. In
contrast, LH subunit mRNAs were not increased and
acute LH release was markedly impaired after the
rapid (7.5 min) or slower (120 and 240 min) pulse
intervals. Equalization of total GnRH dose/48 h using
the 7.5- and 240-min intervals did not increase LH
subunit mRNAs to levels produced by the optimal
30-min interval. These data indicate that the frequency of the pulsatile GnRH stimulus regulates
expression of a and LH/? mRNAs in male rats. Further, GnRH pulse frequencies that increase subunit
mRNA concentrations are associated with continuing LH responsiveness to GnRH. (Molecular Endocrinology 1: 834-838, 1987)
pulsatile manner (4, 7) resulting in intermittent LH release (4, 6, 8-10). The physiological importance of a
pulsatile GnRH signal was elegantly demonstrated in
the monkey by Belchetz et a/. (11), who showed that
GnRH pulses were required to maintain LH release and
that a continuous GnRH infusion desensitized LH secretion. This finding has been confirmed in rats and
sheep (12-14). Desensitization of LH secretion requires
GnRH interaction with its receptor (15), but cannot be
explained by altered receptor number of affinity (16)
and appears to result from intracellular events consequent upon continued occupancy of the receptor by
GnRH or a GnRH agonist. The intracellular mechanisms
which are responsive to a pulsatile GnRH signal are
known, but the requirement for an intermittent stimulus
to initiate and maintain LH synthesis and secretion is
well documented in several species including man (17).
Alterations in the frequency of GnRH secretion occur
in various physiological states, suggesting that changes
in the patterns of GnRH secretion may be important in
regulating gonadotropin secretion. For example, GnRH
pulse frequency increases during the latter part of the
follicular phase of the menstrual cycle in primates (1820) and preceding the preovulatory LH surge in sheep
(21). GnRH pulse frequency increases during sexual
maturation in rats (22) and during the nocturnal increase
in LH secretion in early pubertal boys (17). Further, the
observed postcastration rise in serum LH is accompanied by an increase in GnRH pulse frequency in both
rats and sheep (8, 10, 23). Thus fast GnRH pulse
frequencies are present at times of increasing or elevated LH secretion. Although no data is available concerning the role played by the GnRH pulsatile pattern
on LH synthesis, it is reasonable to suggest that an
increase in GnRH frequency may be important in stimulating LH synthetic mechanisms which would be required to maintain high levels of LH secretion.
The present study was conducted to examine the
influence of GnRH pulse frequency on LH subunit gene
INTRODUCTION
LH is a pituitary glycoprotein composed of two subunits
(a and j3), which are coded by separate genes (1-3).
GnRH regulates LH synthesis and secretion (4-6) and
studies in several mammalian species have shown that
GnRH is released into the hypophysial-portal blood in a
0888-8809/87/0834-0838$02.00/0
Molecular Endocrinology
Copyright © 1987 by The Endocrine Society
834
GnRH Regulation of LH Subunit mRNAs
expression as assessed by changes in a and LH/3
mRNA concentrations. The animal model used was a
castrate male rat in which a physiological concentration
of serum testosterone (T) was maintained by the sc
insertion of silastic implants containing T. Selection of
this model was based on our earlier studies (24), which
showed that T replacement inhibited the postcastration
rise in serum LH and pituitary GnRH receptors in male
rats in a similar manner to the effects of anti-GnRH sera
and hypothalamic lesions (25). Also, Steiner et al. (10)
showed that LH pulses occurred infrequently, less than
4/24 h, or were totally eliminated by T implants in
castrate male rats. Thus, it appears that constant serum
T concentrations inhibit GnRH pulsatile secretion (10)
and that the castrate testosterone-replaced (T-replaced) animal is relatively GnRH deficient. This model
allows examination of the effects of exogenous GnRH
administration in a situation which is not unduly complicated by endogenous GnRH secretion.
835
LH SUBUNIT mRNA RESPONSES TO GnRH PULSE FREQUENCY
Castrate
600 -,
500-
150-
C
100-
SERUM LH
after last GnRH
pulse
o basal
• peak (20 min)
50-
0500400-
1
ALPHA mRNA
m
LH BETA mRNA
300-
<
Q
09
200-
I 10°|
T replacement at the time of castration prevented the
postcastration rise in serum LH, a and LH/3 subunit
mRNA concentrations (Fig. 1). After 48 h of treatment,
a significant acute LH response to the last GnRH pulse
was only observed after pulse intervals of 15, 30, and
60 min, compared to T-replaced controls receiving saline injections. Rats that received faster (7.5 min) or
slower (120 and 240 min) GnRH pulses did not demonstrate acute LH release to the last GnRH bolus, aSubunit mRNA concentrations in T-replaced controls
were 109 ± 23 pg cDNA bound/100 ng pituitary DNA.
The administration of GnRH at intervals between 15
and 60 min stimulated a 3- to 4-fold increase after 48
h, with maximal values (429 ± 23 pg cDNA/100 ^g
DNA) seen in the 30-min interval group. aimRNA was
not elevated after the more rapid or slower GnRH
frequencies. The 30 min GnRH pulse interval stimulated
a 3-fold increase in LH/3 mRNA (98 ± 7 vs. 30 ± 5 pg
cDNA bound/100 ^g pituitary DNA in saline-injected
controls). LH/3 mRNA concentrations were also increased in rats receiving 15- and 60-min GnRH pulses,
but values after the 30-min pulse regimen were higher
(P < 0.01) than all other GnRH-treated groups.
We assessed whether the stimulatory effect of the
30-min pulses on LH subunit mRNAs was a function of
GnRH frequency and/or reflected the total GnRH dose
given over the experimental duration. The effects of 30min GnRH pulses were compared to the fastest (7.5
min) and the slowest (240 min) intervals such that the
GnRH pulse doses were adjusted to equalize the total
dose given over 48 h (Fig. 2). Despite the equalization
of total GnRH dose, the rapid 7.5-min pulse interval
was ineffective in stimulating acute LH release and LH
subunit mRNA concentrations. Increasing the dose of
GnRH in the 240-min pulse interval group from 25 to
200 ng did result in LH release to the last GnRH pulse
and a and LH/3 mRNAs were elevated (P < 0.05 or
0.01 compared to T-replaced controls). However, LH
I
200-
_
^1
RESULTS
Castrate + T implant
0100-
Z
Q
O
80-
g
60
m±SE
• p<0.01 v. saline
"
4020-
30
0-
Saline
7.5 15 30 60120 240 min Pulse Interval
GnRH 25 ng/pulse
Fig. 1. Effects of the Frequency of GnRH Pulses on LH Subunit
mRNA
Concentrations and LH Secretion. Serum LH values before
(O) and 20 min after (peak) the last GnRH pulse are shown in
the top panel, a and LH/3 mRNA concentrations are shown in
the middle and lower panels, respectively. Saline pulses were
given at 30-min intervals for 48 h to castrate (far left panel)
and T-replaced control animals. Each point represents mean
(m) ± SE, n = five to 12 rats per group; *, P < 0.01 vs. salinepulsed, castrate + T controls.
subunit mRNAs were still lower (P < 0.01) than concentrations seen after 30-min GnRH pulses (25 ng).
Thus, the optimal stimulatory effect of the 30-min GnRH
pulse interval appears to involve intracellular mechanisms which respond to frequency per se and do not
reflect the effects of a specific dose of GnRH over the
48 h of the study.
DISCUSSION
Other hypothalamic neuropeptides such as TRH (26,
27) and GHRH (28) have been shown to stimulate
pituitary hormone gene expression. We recently
showed that GnRH stimulated an increase in LH subunit
mRNA concentrations in male rats (29). These findings
complement earlier studies which examined the role of
GnRH in regulating LH synthesis and showed that
GnRH increases subunit translation and glycosylation
(30, 31). As previously noted (29) pituitary concentra-
Vol 1 No. 11
MOL ENDO-1987
836
EFFECT OF GnRH DOSE AND PULSE INTERVAL
ON LH SUBUNIT mRNAs
Castrate
Castrate + T implants
SERUM LH
after last GnRH
pulse
I I basal
ALPHA mRNA
LH BETA mRNA
m + SE
• p = 0.01 v. saline
•* p 0.05 v. saline
Sal Sal
30 30
25
30
2.4
6.6
7.5
2.4
200 ng/pulse
240 min pulse interval
2.4 Total GnRH dose
(ug/48h)
Fig. 2. Effects of GnRH Dose and Pulse Interval on LH Subunit
mRNAs and LH Release
The 30-min pulse interval (25 ng/pulse) was compared to
the fastest (7.5 min) and the slowest (240 min) treatment
intervals, such that the dose of GnRH/pulse was adjusted to
equalize the total GnRH dose given over 48 h (2.4 ug). Castrate
(far left panel) and T-replaced controls received saline (sal)
pulses at 30-min intervals for 48 h. The mean (m) ± SE for
each group is shown; n = five to 12 rats per group; **, P <
0.05; *P, <0.01 vs. castrate + T controls.
tions of amRNAs are 3- to 4-fold greater than levels of
LH/3. This is expected in view of the a-subunit being
common to LH, FSH, and TSH. Recent evidence suggests that /5-subunit gene expression may be a limiting
factor in the synthesis of pituitary glycoprotein hormones (32-34). For example, TSH/3 and LH/3 mRNAs
are more sensitive that amRNA to negative feedback
regulation by T4 and gonadal steroids. Papavasiliou et
al. (29) made similar observations in regard to the
stimulation of LH subunit mRNAs. In T-replaced male
rats a narrow range of GnRH dose per pulse was
required to stimulate LH/? mRNA, but all GnRH pulse
doses increased mRNA. In the present study the frequency of GnRH stimulation did not exert differential
effects on a and LH/3 mRNAs. Both subunit mRNAs
were increased to similar degrees by the same range
of GnRH pulse frequencies.
Although evidence for direct pituitary effects of T on
LH secretory function have been presented (35), its
major action appears to be inhibition of GnRH secretion
from the median eminence. Castration results in an
increase in GnRH pulse frequency (10, 23), up-regulation of pituitary GnRH receptors (24, 25, 36), elevated
LH subunit mRNA concentrations (34, 37), increased
LH subunit translation (38), and increased pituitary LH
content and release (34, 37). Recent data from our
laboratory suggest that the rise in GnRH pulse frequency after castration may play an important role in
the increased gonadotrope secretory activity. Katt et
al. (36) demonstrated that a 30-min pulse interval (25
ng/pulse) was optimal in stimulating GnRH receptor upregulation and resulted in similar receptor concentrations to those present in castrate male rats. Further,
the present study (Fig. 1) revealed that 30-min pulses
stimulated an increase in « and LH/3 mRNAs to concentrations similar to those in male rats 48 h after castration. These findings appear to be physiologically significant since GnRH pulse intervals decrease from 145
min to 20-30 min in male rats after gonadectomy (10).
Since our previous study (36) found that GnRH pulse
intervals of 7.5-120 min stimulated elevations in GnRH
receptors, the present data imply that the LH synthetic
and secretory responses to GnRH pulse frequency may
be more selective than GnRH receptor regulation.
LH subunit mRNA concentrations are also known to
change during reproductive cycles in female animals, a
and LH/3 mRNAs increase during the preovulatory LH
surge in sheep (39, 40). During the rat estrous cycle
(41) both LH subunit mRNAs increase on diestrus (the
day before the preovulatory LH surge), but only LH/3
mRNA is transiently increased 3-fold before the LH
surge on the afternoon of proestrus. The selective
increase in LH/3 mRNA may not be a prerequisite for
the LH surge, as we recently showed that only asubunit mRNA levels were elevated during the afternoon LH surge in ovariectomized, estradiol-treated rats
(42). Ovarian steroids are known to influence LH secretion by actions directly on the pituitary (43) and by
altering hypothalamic GnRH secretion (44). Further,
variations in GnRH pulse frequency have been described during the reproductive cycles of female rats
and sheep (45, 46). Thus the observed differences in
LH subunit gene expression seen in the above physiologic models may result from a combination of the
effects of alterations in the patterns of GnRH pulsatile
secretion and the prevailing ovarian steroid milieu.
Neuroendocrine regulation of anterior pituitary secretion involves intermittent secretion of releasing or inhibiting hormones and adrenocorticotropin, TSH, GH, and
PRL are secreted in a pulsatile manner (47, 48). The
pulsatile nature of GnRH-LH secretion appears to be
common to all species including human (17) and several
lines of evidence suggest that the ability to change
GnRH pulse frequency is an important factor in the
regulation of reproductive function in primates. LH pulse
frequency, and by inference GnRH pulse frequency,
increases during the mid- to late follicular phase of the
human menstrual cycle (18). In GnRH deficient monkeys, GnRH pulses given every 3 h did not stimulate
ovulation, whereas GnRH pulses given every 60-90
min were consistently effective (49, 50). LH pulse frequency is markedly reduced in women with anovulation
due to hypothalamic amenorrhea (51,52), and ovulation
can be induced by exogenous GnRH pulses given every
60-90 min. These studies imply that the ability to
GnRH Regulation of LH Subunit mRNAs
secrete GnRH pulses at a fast frequency may be important for the expression of an LH surge in some
mammalian species. Further, the present data confirm
earlier studies on the effects of GnRH pulse frequency
on LH secretion and suggest that a narrow window of
GnRH frequency is required to maintain maximal LH
synthetic capacity. Finally, our data do not address the
mechanisms of GnRH-induced elevations in a and LH/3
mRNAs. However, GnRH stimulation of LH subunit
polypeptide chain synthesis is inhibited by actinomycin
D (30), suggesting that GnRH stimulates transcription
of the LH subunit genes in a manner analogous to the
actions of TRH and GHRH.
In conclusion, the results indicate that the frequency
of the pulsatile GnRH stimulus can regulate both LH
subunit mRNAs and LH secretion in male rats, with
frequencies between 15 and 60 min being maximally
effective. Further, the data suggest that the changes in
GnRH pulse frequency which occur after castration and
during the estrous cycle may be important in regulating
LH synthesis and release.
MATERIALS AND METHODS
Adult male Holtzman rats were castrated and two 20-mm
silastic implants containing T were inserted sc to produce a
serum T concentration of 2.3 ± 0.12 ng/ml (mean ± SE). Empty
implants were inserted into castrate control rats. After recovery from anesthesia, animals were placed into restraint cages
and given GnRH (25 ng/pulse) at intervals between 7.5 and
240 min via a carotid cannula attached to an automatic pump
(Autosyringe AS2C) for 48 h. Control animals received saline
pulses at 30-min intervals for 48 h. Acute LH release was
measured after 48 h of treatment by obtaining serum samples
before (basal) and 20 min after (peak) the last GnRH injection.
Anterior pituitaries were removed under sterile conditions,
homogenized in 10 mM Tris, 0.5% Nonidet P-40,1 IDM EDTA
(pH 7.4), and the homogenate was centrifuged at 13,000 x g
for 5 min. Nuclear pellets were sonicated and DNA measured
by fluorometric assay (53). Total cytoplasmic RNA was extracted using a phenol-chloroform-isoamyl alcohol mixture
(100:100:1) and measured by absorbance at 260 nm. a and
LH(8 mRNA concentrations were determined by dot blot hybridization assay (37) using saturating amounts of 32P-labeled
a and LH/3 cDNA probes (SA, 2-5 x 108 cpm/^g cDNA).
Messenger RNA concentrations are expressed as picograms
of cDNA bound per 100 ng pituitary DNA. Serum samples
were measured for LH and T by RIA.
The data was analyzed by one-way analysis of variance and
differences between experimental groups determined by Duncan's multiple range test.
Acknowledgments
The authors would like to thank Dr. W. W. Chin who kindly
provided the original a and LH/3 cDNA probes. We also thank
Pamela Dotimas and Linda McCrate for their assistance in
preparing this manuscript.
Received June 24,1987. Accepted September 11,1987.
Address requests for reprints to: J. C. Marshall, Division of
Endocrinology and Metabolism, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109.
These studies were supported by USPHS Grant HD-11489
(to J.C.M.) and by NRSA Fellowship HD-7027 (to D.J.H.).
837
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