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Original Studies |
, a Puberty-Inducing Growth Factor, But Not Luteinizing Hormone-Releasing Hormone Neurons1
Division of Neuroscience (H.J., M.E.C., A.C., Y.J.M., S.R.O.), Oregon Regional Primate Research Center/Oregon Health Sciences University, Beaverton, Oregon, 97006; Clinics of Pediatrics (H.J., K.H.P.B.) and Neurosurgery (M.W.), University Hospital Hamburg-Eppendorf, 20246 Hamburg, Germany; Center for Neurological Surgery (P.C.) and Division of Pediatric Endocrinology (M.S.S.), UMDNJ, New Jersey Medical School, Newark, New Jersey 07103-2499; Department of Anatomy and Cell Biology (J.W.W.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; and Departments of Neurosurgery and Pediatrics (J.H.P.), Oregon Health Sciences University, Portland, Oregon 97201-3098
Address correspondence and requests for reprints to: Sergio R. Ojeda, Division of Neuroscience/Oregon Regional Primate Research Center, Oregon Health Sciences University, 505 NW 185th Avenue, Beaverton, Oregon 97006.
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Abstract |
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(TGF), has been
implicated as a facilitatory component of the glia-to-neuron signaling
process controlling the onset of female puberty in rodents and nonhuman
primates. Hypothalamic hamartomas (HH) are tumors frequently associated
with precocious puberty in humans. The detection of LHRH-containing
neurons in some hamartomas has led to the concept that hamartomas
advance puberty because they contain an ectopic LHRH pulse generator.
Examination of two HH associated with female sexual precocity revealed
that neither tumor had LHRH neurons, but both contained astroglial
cells expressing TGF and its receptor. Thus, some HH may induce
precocious puberty, not by secreting LHRH, but via the production of
trophic factors—such as TGF—able to activate the normal LHRH
neuronal network in the patient’s hypothalamus. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Recent studies in rodents and nonhuman primates indicate that the
onset of puberty is not only influenced by neuronal networks, but also
by astroglial cells, which exert their effects via the secretion of
trophic polypeptides (for a review see Ref. 13). Prominent among these
substances is transforming growth factor (TGF), a member of the
epidermal growth factor (EGF) family shown to be involved in both the
hypothalamic control of normal puberty (14) and sexual precocity
induced by hypothalamic lesions (15). When cells genetically modified
to overexpress the human TGF gene were grafted near LHRH nerve
terminals in the rat hypothalamus, they caused sexual precocity (16),
indicating that a focal increase in TGF secretion suffices to
activate LHRH release and initiate the pubertal process in otherwise
normal animals. We now show that some HH do not contain LHRH neurons,
but are instead endowed with TGF-producing astroglial cells. Thus,
like astrocytes of the normal hypothalamus in lower species, HH may use
TGF-dependent signaling pathways for the precocious activation of
pubertal LHRH release in humans.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Case 1
This patient exhibited signs of sexual precocity at 2.5 yr of
age, including breast development (Tanner stage IV), pubic hair
progression (stage II), and vaginal bleeding. By 4.5 yr of age she had
grown beyond the 97th percentile. No epileptic activity or gelastic
seizures were detected. Serum LH, FSH, and estradiol (E2)
levels were elevated (Table 1
). Abdominal
ultrasound showed an enlarged uterus and ovaries containing follicular
cysts. A cranial magnetic resonance image (MRI) revealed a large
nonenhancing, relatively isointense hypothalamic mass encroaching on
the retrosellar cistern (Fig. 1, A and
B). A hypertrophic pituitary gland was
also observed (Fig. 1B). Right frontal craniotomy performed at 4 yr, 10
months of age revealed a white-to-gray hypothalamic mass with an
abnormal vascular pattern located between the lateral border of the
chiasm and the medial border of carotid artery (Fig. 1A). The tumor was
diagnosed as a hamartoma. Three months after its subtotal resection,
treatment with leuprolide acetate (Lupron) had to be
initiated because of an elevated LH and FSH response to LHRH
stimulation. Five months later, the dose of the analog was increased
from 11.25 to 15 mg/month because the LH response to LHRH stimulation
was still high. MRI scanning demonstrated the persistence of an
isointense nodular mass close to the tuber cinereum, but without
compromising the optic chiasm. The treatment with Lupron was
discontinued 1 yr later because the patient complained of severe
headaches. After a short period of treatment with Histralin (another
LHRH analog), therapy with Nafrelin instituted 9 months
later successfully inhibited the gonadotropin response to LHRH (Table 1) and the progression of puberty.
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Case 2
The second patient started monthly vaginal bleeding at 4 months
of age. At the time of diagnosis (9 months of age), abdominal
ultrasound revealed an enlarged uterus and ovaries with follicular
cysts. Bone age was advanced to 2 yr. Serum LH, FSH (basal and after
LHRH challenge), and E2 levels were elevated (Table 1
).
Breast development had progressed to Tanner stage II, but no pubic hair
had yet appeared. MRI analysis revealed a slightly heterogenous mass
broadly attached to the hypothalamus and bulging into the third
ventricle (Fig. 1C). Biopsy of the tumor performed at 1 yr of age
showed the presence of normal glia and neuronal cells, as well as some
reactive astrocytes, consistent with the features of a hamartoma.
Treatment with the LHRH analog Decapeptyl Depot
(Triptorelin acetate), caused complete gonadotropin and E2
suppression (Table 1) and remission of pubertal development. Bone age
progression continued during the first years of treatment (11 yr of
bone age at 6 yr of chronological age), as observed by others during
LHRH therapy (18), decreasing after the patient was 6 yr old. At 9 yr
of age the ratio between 
bone age to chronological age
decreased from 1.73 to 0.5, improving the predicted adult height from
145 to 160 cm.
Consistent pathological electroencephalographic signs were first seen
at 18 months of age, paralleling the appearance of gelastic seizures.
Despite intensified antiepileptic therapy, the seizure activity became
more prominent. The electroencephalographic appearance eventually
showed bilateral synchronized hyperexcitability and frontal monomorphic
rhythms, making removal of the hamartoma necessary. Surgery was
performed when the patient was 9.6 yr of age. Plasma levels of TSH,
ACTH, insulin-like growth factor-I, and the binding protein IGF-BP3
remained at normal values before and after the operation. Postsurgery
MRI showed the pituitary stalk shifted ventrally, the tumor cavity
filled with cerebrospinal fluid, and a small residual tumor mass
located dorsally near the crura cerebri (Fig. 1D). Seizure activity
decreased but did not disappear despite the anticonvulsive therapy,
with the electroencephalographic appearance changing to focal
hyperactivity in the left frontal parietal region. Treatment with the
LHRH analog was terminated 9 months after surgery, a time at which an
adult height of 160 cm could reliably be predicted.
LHRH and TGF immunohistochemistry
Tumor specimens from Case 1 were embedded in paraffin and then
serially sectioned at 4 µm. Specimens from two cerebellar
astrocytomas (one from a 6-yr-old male and one from a 16-yr-old female)
were also processed to determine if the TGF gene is expressed in
transformed astrocytes. Before staining, the sections were
deparaffinized and treated with target unmasking fluid (Signet
Laboratories, Dedham, MA) to reactivate epitopes that may be
masked by the paraffin embedding. Tumor specimens from Case 2 were
immersed in Zamboni’s fixative after surgery, fixed overnight at 4 C,
and stored in phosphate-buffered saline at 4 C until further
processing.
LHRH-containing cells were identified with either a monoclonal antibody
(HU4H3, 1:2,000) that detects only the mature LHRH decapeptide (19) or
with polyclonal antibody ARK-2 (1:5,000), shown to detect the LHRH
precursor in primate brain (20). Astrocytes were identified by their
content of glial fibrillary acidic protein (GFAP) using either a
polyclonal antibody (R77, 1:2,000; a gift from L. Eng, Stanford
University, Palo Alto, CA) or a monoclonal antibody (Sigma Chemical
Co., St. Louis, MO; 1:20,000). TGF- was detected as described (15),
with a polyclonal antibody (1:1,500) that recognizes the intracellular
domain of the human TGF- precursor protein (21). EGF receptors
(EGFR), which bind both EGF and TGF, were detected with polyclonal
antibody RK-2 (1:1,000), which recognizes a peptide sequence in the
C-terminus of human EGFR (22).
For single staining light microscopy immunohistochemistry, LHRH and
GFAP immunoreactions were developed to a brown color with the chromogen
3'3-diaminobenzidine tetrahydrochloride (DAB, Sigma Chemical Co.). For
double staining, the sections were first processed for GFAP detection
and then were incubated with either TGF or EGFR antibodies, followed
by development of the reactions with the chromogen benzidine
dihydrochloride (Sigma Chemical Co.) to a blue color (23), as described
(24). Double immunohistofluorescence was performed using the same
primary antibodies described above, followed by development of the
immunoreactive reactions with fluorochrome-conjugated secondary
antibodies. Cell nuclei were stained with the vital dye Hoechst
(Molecular Probes, Eugene, OR; 1 min with a 1 µg/mL solution).
Cryostat (10 µm) sections from an adult female rhesus monkey
hypothalamus were used to show the ability of the monoclonal antibody
to LHRH to detect LHRH neurons in a species highly related to humans.
Controls consisted of sections incubated without the primary
antibodies.
Confocal imaging
Images were acquired using a Leica TCS NT confocal system with a 40x NA1.25 PL APO objective. Hoechst was excited with the 367-nm line of an ArUV laser and detected through a 440 ± 40-nm bandpass filter. FITC and Texas Red were imaged simultaneously, using the 488-nm and 568-nm lines of an Ar and Kr gas laser, respectively, for excitation, a double dichroic at 488 nm/568 nm, and a reflective mirror for wavelengths less than 580 nm in front of the first detection channel. A bandpass emission filter of 530 nm was used for FITC and a long pass filter at 590 nm for Texas Red. The intensity of the excitation light in each channel was adjusted so that the contribution of fluorescein to light detected in the Texas Red channel was negligible. Typically, eight optical sections 1 µm apart were acquired for each image. Colors were merged, and sections were projected into a single plane using MetaMorph (Universal Imaging, West Chester, PA). Images were further processed using Photoshop 4.0 (Adobe Systems, San Jose, CA).
In situ hybridization
A specimen of the HH from Case 1 was processed for hybridization
histochemistry (25). Cells containing TGF messenger RNA (mRNA) were
detected with a 35S-UTP-labeled complementary RNA,
complementary to a 316-nucleotide segment contained in the coding
region of human TGF mRNA (26). Control sections were hybridized with
a sense RNA synthesized from the same DNA template, but in the opposite
direction.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Routine histology. Frozen sections revealed the presence of scattered, large neurons and reactive astrocytes. The neurons appeared irregular in size and were irregularly clustered.
Immunohistochemistry. No LHRH neurons were detected in serial
sections of two specimens from the HH (Fig. 2A). In contrast, a few neurons contained
TGF immunoreactive material (Fig. 2B). TGF immunoreactivity was,
however, more generalized in astrocyte-like cells (Fig. 2C). Double
immunohistochemistry demonstrated the widespread expression of TGF
(Fig. 2D, punctated dark blue staining denoted by arrows) in
cells identified as astrocytes by their content of GFAP (Fig. 2D, smooth brown staining). A similar colocalization was observed in the
two cerebellar astrocytomas (Fig. 2F). In this case, however, the
tumors were almost exclusively composed of astrocytes (smooth brown
color), containing abundant TGF immunoreactivity (punctated dark
blue staining shown by arrows). As seen in the normal
hypothalamus of other species (24, 27), HH astrocytes also contained
EGFR immunoreactivity (Fig. 2E, dark blue grains shown by
arrows). Tissue sections incubated without the primary
antibodies did not show cellular staining over background levels (data
not shown). The immunohistochemical specificity of each of the
antibodies used has been previously reported previously in detail (15, 19, 28).
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mRNA was abundant in
cells containing small, darkly stained nuclei characteristic of
astroglial cells (Fig. 3).
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Routine histology/immunohistochemistry. As in Case 1, this tumor was composed of phenotypically normal neurons and astrocytes. No proliferation was detected by staining with the proliferation marker MIB1 (29).
LHRH and TGF immunohistochemistry. No cells or fibers
containing the mature LHRH decapeptide (Fig. 4A) or its precursor (data not shown)
were detected in serial sections of specimens from this tumor. The same
antibodies that failed to detect cells containing the mature LHRH
peptide or the LHRH precursor in the HH readily detected such cells and
LHRH fibers in the rhesus monkey hypothalamus (Fig. 4, B and C, and
Ref. 20 , respectively). In contrast to this absence of LHRH neurons,
the tumor showed an extensive astrocytic network (Fig. 5A), containing TGF (Fig. 5, B and C).
A higher magnification view of the tissue showed that the content of
TGF protein varies extensively among astrocytes and within different
portions of the same cell (Fig. 6).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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, a growth factor implicated as a
facilitatory component of the pubertal process in rodents and nonhuman
primates (14, 27). Surgical resection of these tumors was performed as
either an alternative to approximately 6 years of treatment with an
LHRH agonist (Case 1) or as a treatment of the patient’s intractable
gelastic/epileptogenic seizures (Case 2). In both cases, TGF was
abundantly expressed in astroglial cells. Importantly, examination of
one of the tumors revealed that the astrocytes also contain EGFR, the
receptor that initiates TGF biological actions. In both rodents and
nonhuman primates, TGF is expressed in astroglial cells of
hypothalamic regions involved in the control of gonadotropin secretion
(14, 27). In both species, hypothalamic TGF gene expression
increases at the time of puberty (14, 27). The increase is more
prominent in the region of the median eminence, where LHRH neurons send
their neurosecretory axons, and in the preoptic area, where many LHRH
neuronal perikarya are located.
Experiments showing that puberty can be advanced or delayed by
manipulating TGF availability to pertinent regions of the
neuroendocrine brain have demonstrated the importance of local changes
in TGF production for the timely initiation of female puberty. Thus,
pharmacological blockade of EGFR directed to the median eminence
of the hypothalamus delayed the onset of puberty in rats (14).
Conversely, cells genetically engineered to produce human TGF
advanced the onset of puberty when grafted either near LHRH nerve
terminals in the median eminence or in the vicinity of the LHRH cell
bodies in the preoptic region (16). Cells grafted away from LHRH
neurons were ineffective, indicating that focal increases in TGF
production must occur near LHRH neurons to accelerate the pace of
female sexual maturation.
The abundance of TGF in astroglial cells of the two HH studied
was striking. Interestingly, most, if not all, astrocytes in the
cerebellar astrocytomas examined displayed abundant TGF
immunoreactivity, indicating that neoplastic transformation of
astroglial cells is not only associated with increased expression of
EGFR (30), but also with an enhanced production of one of its ligands.
Despite their TGF content, neither cerebellar tumor was associated
with precocious puberty, reinforcing the concept (16) that only a close
proximity of TGF-producing cells to the LHRH neuronal network allows
the TGF-dependent activation of LHRH secretion.
The present study does not contest the current concept that some HH
induce sexual precocity because they contain an ectopic LHRH "pulse
generator." Instead, our results document the existence of HH that
lack LHRH neurons but contain TGF, a growth factor shown to hasten
the pubertal process when made available to LHRH neurons. Other authors
have previously described the absence of LHRH in HH associated with
precocious puberty (31, 32). It is still possible that the presence of
few scattered LHRH neurons may have escaped detection in these studies,
including our own.
Resembling normal hypothalamic astrocytes (24, 27), HH astrocytes also
contain EGFR and, thus, can respond to TGF with the release of
prostaglandin E2 and other bioactive substances, able to
act directly on LHRH neurons to promote LHRH secretion (33). Whereas in
the normal hypothalamus LHRH neurons and astrocytes are in intimate
contact, bioactive substances released by HH astrocytes in response to
TGF would be expected to act on LHRH neurons located in the
adjacent, normal hypothalamic tissue. In addition, the tumor may
secrete TGF itself, which by reaching responsive, EGFR-bearing
hypothalamic astrocytes may contribute to further stimulate the LHRH
neuronal network of the patient’s hypothalamus. Such a secretory
capacity may contribute to explaining the puberty-inducing capacity of
small-size HH lacking myelinated fibers connecting the tumor to
surrounding hypothalamic regions (2), as well as the ability of HH
almost devoid of neuronal elements to induce sexual precocity (34).
Regardless of the alternative or complementary mechanisms that may
underlie the ability of HH to accelerate sexual development, the
present results indicate that glial substances, such as TGF, found
to be involved in facilitating the normal process of puberty, may also
play an important role in the physiopathology of HH-induced
sexual precocity. In a broader sense, they emphasize the possibility
that circumscribed derangements in hypothalamic glial activity may
contribute to idiopathic precocious puberty of central origin.
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Footnotes |
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Received May 27, 1999.
Revised July 27, 1999.
Accepted August 19, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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(TGF) gene expression in the
hypothalamus is developmentally regulated and linked to sexual
maturation. Neuron. 9:657–670.[CrossRef][Medline]
contributes to the mechanism by which
hypothalamic injury induces precocious puberty. Proc Natl Acad Sci USA. 88:9743–9747. expression to discrete loci of
the neuroendocrine brain induces female sexual precocity. Proc Natl
Acad Sci USA. 94:2735–2740.
precursor protein in transfected mammalian cells. Mol Cell Biol. 7:1585–1591.
overexpression in transgenic mice induces liver neoplasia and abnormal
development of the mammary gland and pancreas. Cell. 61:1137–1146.[CrossRef][Medline]
(TGF) and its receptor in the hypothalamus of female rhesus
macaques. Neuroendocrinology. 60:346–359.[Medline]
with
secretion of neuroactive substances that stimulate the release of
luteinizing hormone-releasing hormone. Endocrinology. 138:19–25.This article has been cited by other articles:
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  B. M. Windsor-Engnell, E. Kasuya, M. Mizuno, K. L. Keen, and E. Terasawa An increase in in vivo release of LHRH and precocious puberty by posterior hypothalamic lesions in female rhesus monkeys (Macaca mulatta) Am J Physiol Endocrinol Metab, April 1, 2007; 292(4): E1000 - E1009. [Abstract] [Full Text] [PDF]
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 S. R. Ojeda, A. Lomniczi, C. Mastronardi, S. Heger, C. Roth, A.-S. Parent, V. Matagne, and A. E. Mungenast Minireview: The Neuroendocrine Regulation of Puberty: Is the Time Ripe for a Systems Biology Approach? Endocrinology, March 1, 2006; 147(3): 1166 - 1174. [Abstract] [Full Text] [PDF]
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 J. L. Freeman, L. T. Coleman, R. M. Wellard, M. J. Kean, J. V. Rosenfeld, G. D. Jackson, S. F. Berkovic, and A. S. Harvey MR Imaging and Spectroscopic Study of Epileptogenic Hypothalamic Hamartomas: Analysis of 72 Cases AJNR Am. J. Neuroradiol., March 1, 2004; 25(3): 450 - 462. [Abstract] [Full Text] [PDF]
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 H. Jung, E. N. Probst, B. P. Hauffa, C.-J. Partsch, and O. Dammann Association of Morphological Characteristics with Precocious Puberty and/or Gelastic Seizures in Hypothalamic Hamartoma J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4590 - 4595. [Abstract] [Full Text] [PDF]
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 S M Ng, Y Kumar, D Cody, C S Smith, M Didi, and M D C Donaldson Cranial MRI scans are indicated in all girls with central precocious puberty * COMMENTARY Arch. Dis. Child., May 1, 2003; 88(5): 414 - 418. [Abstract] [Full Text] [PDF]
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 V. Prevot, C. Rio, G. J. Cho, A. Lomniczi, S. Heger, C. M. Neville, N. A. Rosenthal, S. R. Ojeda, and G. Corfas Normal Female Sexual Development Requires Neuregulin-erbB Receptor Signaling in Hypothalamic Astrocytes J. Neurosci., January 1, 2003; 23(1): 230 - 239. [Abstract] [Full Text] [PDF]
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 L. Abdennebi, E. Y. Chun, H. Jammes, D. Wei, and J. J. Remy Maintenance of Sexual Immaturity in Male Mice and Bucks by Immunization Against N-Terminal Peptides of the Follicle-Stimulating Hormone Receptor Biol Reprod, January 1, 2003; 68(1): 323 - 327. [Abstract] [Full Text] [PDF]
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 R. A. DeFazio, S. Heger, S. R. Ojeda, and S. M. Moenter Activation of A-Type {gamma}-Aminobutyric Acid Receptors Excites Gonadotropin-Releasing Hormone Neurons Mol. Endocrinol., December 1, 2002; 16(12): 2872 - 2891. [Abstract] [Full Text] [PDF]
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M. R. Palmert and P. A. Boepple Variation in the Timing of Puberty: Clinical Spectrum and Genetic Investigation J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2364 - 2368. [Abstract] [Full Text] [PDF]
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