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Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Address all correspondence and requests for reprints to: Dr. Tony M. Plant, University of Pittsburgh, Department of Cell Biology and Physiology, S-330, Biomedical Science Tower, Pittsburgh, Pennsylvania 15261. E-mail: plant1+{at}pitt.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid (GABA) synthesizing enzymes,
glutamate decarboxylase (GAD)65 and GAD67 and
transforming growth factor-
(TGF) in the hypothalamus are
correlated with the arrest of pulsatile GnRH release during infancy in
the agonadal male monkey. This experiment also provided the opportunity
to examine changes in hypothalamic GnRH gene expression during this
critical phase of primate development. Male rhesus monkeys were
castrated at 1 week of age: four were killed 4–7 weeks after
orchidectomy while pulsatile GnRH release was robust as reflected by
high circulating LH levels, and four were killed at 12–15 months of
age after establishing that pulsatile GnRH release had been arrested.
GAD65, GAD67, TGF, and GnRH mRNA levels were
estimated using RNase protection assays employing homologous probes and
the results were expressed relative to cyclophilin mRNA levels. GnRH
peptide was measured by RIA. GAD65 and GAD67
mRNA levels in the hypothalamus of juveniles were significantly greater
than those in neonatal monkeys. On the other hand, hypothalamic TGF
and GnRH mRNA (and peptide) levels in agonadal neonate and juvenile
monkeys were indistinguishable. These results indicate that the
molecular concomitants associated with bringing the hypothalamic GnRH
pulse generator into check in agonadal neonatal males are not a mirror
image of those previously reported at the time this neuronal network is
reactivated at puberty when TGF and GnRH gene expression increase
and GAD65 and GAD67 mRNA levels remain
unchanged. Thus, the neurobiological mechanism that reactivates
pulsatile GnRH release at puberty is likely to involve more than a
simple reversal of that underlying inhibition of the same network in
late infancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypothalamic GnRH stimulation of the pituitary gonadotrophs of the infantile primate, however, is not sustained and gonadotropin secretion later declines guaranteeing a delay in the onset of puberty, which in these species is characteristically protracted (5). Thus, the mechanism that brings pulsatile GnRH release into check during the neonatal-juvenile transition must be viewed as a fundamental component of the control system that times the onset of primate puberty. While the neurobiology of the reactivation of pulsatile GnRH release at the end of the juvenile phase of development has been examined extensively (6, 7, 8, 9), that associated with restraining pulsatile GnRH release during the neonatal-juvenile transition has received little attention.
The idea that the mechanism responsible for imposing the brake on
pulsatile GnRH release in infancy is thrown into reverse at the
termination of the juvenile phase of development to trigger the onset
of puberty is the most parsimonious explanation to account for the time
course of GnRH release from birth until puberty in primates. With this
in mind, hypothalamic levels of the messenger RNAs (mRNAs) encoding
transforming growth factor- (TGF) and the -aminobutyric
acid (GABA) synthesizing enzymes, glutamate decarboxylase
(GAD)65, and GAD67, were
compared in agonadal monkeys during the neonatal (robust GnRH pulse
generator activity) and juvenile (arrested GnRH pulse generator
activity) phase of development. TGF and GABA have been implicated in
the postnatal regulation of GnRH in primates (7, 8, 9) and, during the
juvenile-pubertal transition, hypothalamic levels of the mRNA encoding
TGF have been shown to increase (7, 10), whereas those encoding the
GADs have been reported not to change during this developmental stage
(10, 11). Additionally, this experiment provided an opportunity to
examine changes in hypothalamic GnRH gene expression that occur as the
GnRH pulse generator is brought into check in late infancy. Male rhesus
monkeys were used because the prepubertal hiatus in pulsatile GnRH
release is most marked in the male (5, 12), and it was reasoned that
hypothalamic concomitants would be correspondingly exaggerated and
therefore easier to identify for the first time in the male. The
agonadal model was employed to eliminate any secondary molecular
changes in the hypothalamus, which in intact animals may be anticipated
to occur in response to the declining levels of testicular steroids
during the neonatal-juvenile transition.
It should be noted that the hypothalamic mRNA and protein extracts from the animals employed in this study were used to examine changes in neuropeptide Y (NPY) expression during the neonatal-juvenile transition. The NPY results comprise a component of a comprehensive study of the role of this neuropeptide in regulating pulsatile GnRH release throughout postnatal development in the male monkey, and have been previously reported (10).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). To monitor circulating LH
concentrations, blood samples (1–3 ml) were collected on the day of
castration and thereafter every other week by femoral venipuncture,
while the monkeys were sedated with ketamine hydrochloride
(approximately 20 mg/kg BW). During neonatal development, the infants
were temporarily taken from their mothers while the latter were sedated
with ketamine hydrochloride. The four monkeys studied as juveniles were
separated from their mothers when 6–9 months of age and housed in
pairs in individual cages located in the same building as their
mothers. These animals were fed once a day with Purina Monkey Chow
(Ralston Purina Co., St. Louis, MO) at approximately
1100 h, and fruit was invariably provided in the afternoon.
Water was provided ad libitum.
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RNA and protein preparation
Total RNA was isolated using RNAzol (RNA STAT-60) followed
sequentially by precipitation with isopropanol, washing with ethanol
and solubilization in DEPC-treated water according to the
manufacturer’s instructions (Tel-Test, Friendswood, TX).
RNA integrity was checked by visualization of ethidium bromide-stained
28S and 18S ribosomal RNA bands after migration on agarose gel, and RNA
concentration was determined by measuring absorbance at 260 nm.
Proteins were precipitated sequentially with ethanol and isopropanol from the organic phase after isolation of RNA, followed by washing with 0.3 M guanidine hydrochloride-95% ethanol, and solubilization in 1% SDS according to the manufacturer’s instructions (Tel-Test, Friendswood, TX). Protein concentration was measured by the Bradford assay (Bio-Rad Laboratories, Inc. Hercules, CA).
Complementary DNAs (cDNAs) and riboprobe synthesis
A 204-bp fragment of monkey GnRH and a 364-bp fragment of monkey
TGF cDNAs (7) were kindly provided by Dr. S. R. Ojeda (Oregon
Regional Primate Research Center, Beaverton, OR). A 382-bp fragment of
monkey GAD65 and a 210-bp fragment of monkey
GAD67 cDNAs (13) were kindly provided by Drs.
T. G. Golos and E. Terasawa (Wisconsin Regional Primate Research
Center, Madison, WI). A 117-bp fragment of rat cyclophilin cDNA (14)
was kindly provided by Dr. J. L. Roberts (Mount Sinai School of
Medicine, New York, NY).
The cDNAs were linearized with EcoRI (GnRH, TGF,
cyclophilin) or BamHI (GAD65,
GAD67), then in vitro transcribed with
SP6 (GnRH, TGF) or T7 (GAD65,
GAD67, cyclophilin) RNA polymerase using a
transcription kit (Ambion, Inc., Austin, TX), to
synthesize 32P UTP-labeled RNA antisense probes.
Sense probes were generated from the same cDNA fragments that were used
to synthesize the respective antisense probes. The cDNAs were
linearized with HindIII (GnRH, TGF) or BamHI
(cyclophilin), then in vitro transcribed with T7 (GnRH,
TGF) or T3 (cyclophilin) RNA polymerase.
RNase protection assay
RNase protection assay was performed as described previously
(15, 16). Briefly, 30 µg of RNA were allowed to hybridize with the
32P-labeled antisense RNA probes in solution at
45C overnight, followed by combined RNase A and T1 digestion of
nonhybridized RNA. TGF and GnRH probes were used simultaneously in
the same assay, as were the probes for GAD65 and
GAD67. Stable hybrids were extracted,
ethanol-precipitated, and then denatured and separated on 6%
polyacrylamide-8 M urea gels. The dried gel was exposed in
a Bio-Rad Laboratories, Inc. (Hercules, CA) CS Molecular
Imaging Screen for 16–22 h, and an image of each gel was acquired by a
Molecular Imager (Model GS-525; Bio-Rad Laboratories, Inc.). Antisense probes with 30 µg transfer RNA were run as
controls and were completely digested by RNase A and T1. In addition,
increasing amounts (0, 5, 10, 20, and 40 pg) of in vitro
transcribed sense GnRH, TGF and cyclophilin mRNAs were hybridized
with the corresponding 32P-labeled RNA antisense
probes. In each case, a linear relationship was observed between mRNA
mass and integrated optical density, quantified by Molecular Analyst
Software (Bio-Rad Laboratories, Inc.) after subtraction of
background. In the case of GAD65 and
GAD67, for technical convenience, standard curves
were not generated. Increasing amounts (up to 80 µg) of hypothalamic
mRNA yielded dose-dependent increases in integrated optical density for
all transcripts studied.
The integrated optical density of the TGF, GnRH,
GAD65, and GAD67 hybrids
were normalized to that of cyclophilin and expressed as relative
optical density. The use of homologous antisense probes (TGF, GnRH,
GAD65, and GAD67) yielded
full-length hybrids, although a weak secondary signal was also detected
in the GAD65 assay. In contrast, the use of a
heterologous antisense probe to assay cyclophilin produced two hybrids
of approximately 65 and 45 bp, respectively. This was presumably the
result of sequences mismatches between the monkey mRNA and the rat
antisense probe. As in an earlier study (16), all relative optical
densities were determined using the 45 bp cyclophilin hybrid.
RIAs
Circulating LH concentrations were estimated by a previously
described RIA (10). GnRH content was measured by RIA using the
antiserum R1245 from Dr. T. Nett (17) at a final dilution of 1:90,000.
Synthetic GnRH from Dr. G. Bialy (NICHHD) was used for both the
radiolabeled antigen and reference standard. The antigen-antibody
complex was precipitated with a goat antirabbit globulin
(Antibodies Inc., Davis, CA) at a final dilution of 1:120. The
ED50 of the assay was 15 ng/tube and the minimal
detectable concentration of GnRH was 0.03 ng/tube. The average
intraassay coefficient of variation was 15%. GnRH levels were
expressed as ng/mg protein.
Numerical analysis
Values were expressed as mean ± SD.
Statistical comparisons between experimental groups were made using the
nonparametric Mann-Whitney U test, and differences were
considered to be significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In both the MBH and POA, the mean relative optical density of
GAD65 and GAD67 mRNA
signals in the juvenile group were approximately 30% greater than
those in the neonatal group (Fig. 2). In
contrast, TGF mRNA levels in both MBH and POA were indistinguishable
in neonate and juvenile animals (Fig. 2). These three transcripts were
detected in the cortex, but developmental differences in
GAD65, GAD67 and TGF
levels were not observed in this brain region (Table 1).
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). Developmental changes in GnRH peptide
content in these brain regions were also unremarkable (Fig. 3). The
GnRH mRNA signal in cortex was weak and was not quantitated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had anticipated that hypothalamic levels of the mRNA encoding GnRH would decrease during the neonatal-juvenile transition, i.e. in a manner to mirror the previously reported increased expression of this gene during the juvenile-pubertal transition in agonadal males (10). On the other hand, hypothalamic GnRH peptide content was not expected to change in accordance with the results of an earlier study (18). Although the latter prediction was confirmed, surprisingly GnRH mRNA levels in the MBH of neonatal and juvenile animals were indistinguishable; a result similar to that previously reported for the intact female monkey (7). Presumably a decrease in GnRH secretion in the neonatal-juvenile transition is achieved in the absence of a change in GnRH gene expression.
The present study also failed to provide evidence for the hypothesis that the mechanisms responsible for imposing the brake on pulsatile GnRH release in infancy are simply thrown into reverse at the termination of the juvenile phase of development to trigger the onset of puberty. Levels of the mRNAs encoding the GABA synthesizing enzymes, GAD65, and GAD67 in both MBH and POA of juvenile monkeys, in which GnRH pulse generator activity had been arrested, were greater than those in neonatal animals exhibiting robust pulsatile GnRH release. This is in contrast to the absence of a change in these mRNA levels previously established using identical methodology during the juvenile-pubertal transition in the agonadal model (10). Changes in the expression of these genes from the early juvenile stage to adulthood have also been shown to be unremarkable in the intact male monkey (11).
In the rat, GABA levels in the MBH and POA increase during the first 15–20 days of postnatal life (19, 20). Moreover, during this phase of development the electrophysiological action of GABA in regions of the rodent CNS including the hypothalamus changes from one that is stimulatory to one that is inhibitory (21, 22), which has been reported to occur as a result of an increase in the expression of the neuronal Cl- extruding K+/Cl- cotransporter, KCC2 (23). While it is likely that this developmental "maturation" in GABA action is recapitulated in the primate brain, the timing of this event in the monkey is unknown and it is therefore not possible to place into comparative perspective the present finding of an increase in hypothalamic GAD expression during the neonatal-juvenile transition. It should be noted, however, that in rat the increase in hypothalamic GAD levels during neonatal development was paralleled by a similar change in the cortex (20). In the present study of the monkey, however, there was no evidence for changes in GAD expression in the cortex during the neonatal-juvenile transition.
In the adult monkey, orchidectomy results within 42 days in a decrease in the levels of the mRNAs encoding both GAD65 and GAD67 in the MBH (16), and therefore it is unlikely that the increased levels of GAD mRNAs in the MBH observed between 2 months and 15 months of age in the agonadal male monkey was due to an insidious effect on GAD expression triggered by removing the testes at 1 week age.
Studies by Terasawa and her colleagues (8) have provided compelling evidence for the view that GABA plays an important role in restraining GnRH release in the juvenile female monkey before menarche. GABA content in perfusates from the MBH of prepubertal female monkeys is greater than that from pubertal females, and inhibition of GABA synthesis or action results in precocious GnRH release and premature menarche and ovulation (8, 9, 13). We have previously argued that this inhibitory GABA tone may not be as robust in the juvenile male (10), and if this is the case it might be predicted that the increase in the hypothalamic levels of the mRNAs encoding the GADs during the neonatal-juvenile transition would be more striking in the female.
As with changes in GAD gene expression during the
neonatal-juvenile transition, those in TGF at this developmental
stage also failed to mirror those that were previously described during
peripubertal development in the agonadal male (10). In the latter
situation, TGF mRNA levels in the MBH in pubertal monkeys are higher
than those in juvenile animals (10). A similar observation has also
been made in the intact female monkey, although in the latter case the
pubertal increase in TGF gene expression was observed in the both
hypothalamic regions (7). In the case of the female, however,
hypothalamic TGF mRNA levels were also elevated during the neonatal
period (7). Because estradiol treatment of ovariectomized adults
castrated prepubertally does not appear to influence TGF gene
expression (15), it seems reasonable to conclude that the developmental
pattern in hypothalamic TGF expression in the female monkey is a
gonadal independent event. The reasons for the apparent sex difference
in the expression of TGF during the neonatal juvenile transition
remains to be established.
In summary, while GnRH and TGF mRNA levels in the
hypothalamus of the agonadal male monkey do not change at the time when
the GnRH pulse generator is being brought into check during the
neonatal-juvenile transition, expression of the genes encoding
GAD65 and GAD67 increase at
this critical developmental stage. These results are to be contrasted
with those demonstrating that during removal of the prepubertal brake
upon the GnRH pulse generator at the time of the juvenile-pubertal
transition, GnRH and TGF mRNA levels increase while those encoding
the GADs do not change (10). Taken together, these findings indicate
that the molecular concomitants associated with bringing the GnRH pulse
generator into check during infancy are not mirror images of those seen
at the time this neuronal network is reactivated at the end of the
juvenile phase of development. Thus, the neurobiological mechanism that
activates the GnRH neuronal network at the onset of puberty is likely
to involve more than a simple reversal of those underlying inhibition
of the same network in late infancy.
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Acknowledgments |
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Footnotes |
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Received April 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aminobutyric acid is an inhibitory neurotransmitter restricting the
release of luteinizing hormone-releasing hormone before the onset of
puberty. Proc Natl Acad Sci USA 91:395–399This article has been cited by other articles:
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  M. Shahab, C. Mastronardi, S. B. Seminara, W. F. Crowley, S. R. Ojeda, and T. M. Plant Increased hypothalamic GPR54 signaling: A potential mechanism for initiation of puberty in primates PNAS, February 8, 2005; 102(6): 2129 - 2134. [Abstract] [Full Text] [PDF]
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 T. M. Plant and M. L. Barker-Gibb Neurobiological mechanisms of puberty in higher primates Hum. Reprod. Update, January 1, 2004; 10(1): 67 - 77. [Abstract] [Full Text] [PDF]
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 J. Hou, B. Li, Z. Yang, N. Fager, and M. Y. J. Ma Altered Gene Activity of Epidermal Growth Factor Receptor (ErbB-1) in the Hypothalamus of Aging Female Rat Is Linked to Abnormal Estrous Cycles Endocrinology, February 1, 2002; 143(2): 577 - 586. [Abstract] [Full Text] [PDF]
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 E. Terasawa and D. L. Fernandez Neurobiological Mechanisms of the Onset of Puberty in Primates Endocr. Rev., February 1, 2001; 22(1): 111 - 151. [Abstract] [Full Text]
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