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The Journal of Clinical Endocrinology & Metabolism
Copyright © 1999 by The Endocrine Society
Vol. 84, No. 12
Printed in U.S.A.
Some Hypothalamic Hamartomas Contain Transforming
Growth Factor a, a Puberty-Inducing Growth Factor,
But Not Luteinizing Hormone-Releasing
Hormone Neurons*
H. JUNG, P. CARMEL, M. S. SCHWARTZ, J. W. WITKIN, K. H. P. BENTELE,
M. WESTPHAL, J. H. PIATT, M. E. COSTA, A. CORNEA, Y. J. MA, AND S. R. OJEDA
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
ABSTRACT
Activation of LH-releasing hormone (LHRH) secretion, essential
for the initiation of puberty, is brought about by the interaction of
neurotransmitters and astroglia-derived substances. One of these
substances, transforming growth factor a (TGFa), 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 con-
cept 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 TGFa and its
receptor. Thus, some HH may induce precocious puberty, not by
secreting LHRH, but via the production of trophic factors—such as
TGFa—able to activate the normal LHRH neuronal network in the
patient’s hypothalamus. (J Clin Endocrinol Metab 84: 4695– 4701,
1999)
H
factor a (TGFa), 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 TGFa gene were grafted near LHRH nerve
terminals in the rat hypothalamus, they caused sexual precocity
(16), indicating that a focal increase in TGFa 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 TGFaproducing astroglial cells. Thus, like astrocytes of the normal
hypothalamus in lower species, HH may use TGFa-dependent
signaling pathways for the precocious activation of pubertal
LHRH release in humans.
AMARTOMAS are congenital, nonneoplastic tumor-like
masses containing mature cells of normal appearance
(1). Hypothalamic hamartomas (HH) frequently result in sexual
precocity (2–5). Some are also associated with epileptic disorders, mainly gelastic seizures (6, 7). A prevailing view concerning the mechanism by which HH induce sexual precocity is that
the tumors contain ectopic LH-releasing hormone (LHRH) secreting cells, which by functioning independently of the normal
LHRH neuronal network lead to premature activation of pulsatile LHRH release and, thus, to precocious puberty (8, 9). This
view stems from the findings that some HH contain LHRH
immunoreactive cells and fibers (9–12).
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
Received May 27, 1999. Revision received July 27, 1999. Accepted
August 19, 1999.
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.
* This research was supported by NIH Grants HD-25123 (to S.R.O.),
RR-00163 for the operation of the Oregon Regional Primate Research
Center, and P30 Population Center Grant HD-18185, and a grant from
the Deutsche Forschungsgemeinsschaft (Ju 296/1-1, 1-2; to H.J.).
Subjects and Methods
Approval to work with hamartoma specimens was granted by the
Oregon Regional Primate Research Center Institution Review Board on
January 9, 1995.
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 con-
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TABLE 1. Plasma hormone levels in two patients with HH associated with precocious puberty
Basal Plasma Hormone Levels Before LHRH Analog Treatment
Patient 1
Patient 2
a
b
FSH (mIU/mL)
LH (mIU/mL)
E2 (pg/mL)
2.8
7.2
1.2
9.2
250
155
Response to LHRH Beforea and After LHRH Analog Treatmentb
LH (mIU/mL)
Before
After
3.5– 4.8
9.5–16.3
1.1
,0.5
FSH (mIU/mL)
Before
After
1.5
5.1
—
,0.5
E2 (pg/mL)
Before
After
430
—
,0.5
—
LHRH challenge performed at 36 months of age (Patient 1) and 18 months of age (Patient 2).
Patient 1 treated with Nafrelin; Patient 2 treated with Decapeptyl Depot (Triptorelin acetate).
FIG. 1. A, Coronal MRI of the brain from patient 1 showing a hypothalamic mass (arrow) diagnosed as a hamartoma. B, Sagittal MRI view
of the same patient showing the hamartoma (arrow) and a hypertrophic pituitary gland bulging through the diaphragm sellae (arrowhead).
C, Sagittal MRI view of the brain from patient 2 showing a large isointense, sessile mass of tissue (arrow) located at the base of the third ventricle
(arrowhead). D, Postoperative sagittal MRI of the same patient showing the cavity remaining after removal of the tumor (arrow) and the presence
of tumor remnants in this cavity (arrowhead).
taining 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
HH AND GROWTH FACTORS
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.
Unexpectedly, however, 2 yr after initiating the treatment with LHRH
analogs, the patient developed GH deficiency, a most unusual turn of
events in these patients (17). A GH stimulation test (arginine/L-Dopa)
done when her height velocity had decreased to 3.1 cm/y demonstrated
a blunted GH response (9.5 ng/dL). She is currently receiving GH
therapy.
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 d bone age to d 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.
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polyclonal antibody (1:1,500) that recognizes the intracellular domain of
the human TGF-a precursor protein (21). EGF receptors (EGFR), which
bind both EGF and TGFa, 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 393-diaminobenzidine tetrahydrochloride (DAB, Sigma
Chemical Co.). For double staining, the sections were first processed for
GFAP detection and then were incubated with either TGFa 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 mg/mL solution). Cryostat (10 mm) 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
403 NA1.25 PL APO objective. Hoechst was excited with the 367-nm line
of an ArUV laser and detected through a 440 6 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 mm 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 TGFa 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 TGFa mRNA (26). Control sections were hybridized with a sense
RNA synthesized from the same DNA template, but in the opposite
direction.
Results
Case 1
LHRH and TGFa immunohistochemistry
Tumor specimens from Case 1 were embedded in paraffin and then
serially sectioned at 4 mm. 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 TGFa 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-a was detected as described (15), with a
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 TGFa immunoreactive
material (Fig. 2B). TGFa immunoreactivity was, however,
more generalized in astrocyte-like cells (Fig. 2C). Double
immunohistochemistry demonstrated the widespread expression of TGFa (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),
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FIG. 2. A–C, Absence of LHRH neurons (A) in the HH from patient 1, and presence of both neurons (B, arrows) and astroglial cells (C, arrows)
containing TGFa in the same HH. D–F, Double immunohistochemistry demonstrating the presence of both TGFa (D) and EGFR immunoreactivity (E) in astrocytes of the HH from patient 1. Astrocytes are identified by their content of GFAP (smooth brown staining). TGFa and EGFR
immunoreactivities are seen as dark blue punctated staining (arrows) overlaying the GFAP reaction. Bars, 10 mm.
containing abundant TGFa 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).
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 TGFa (Fig. 5, B and
C). A higher magnification view of the tissue showed that the
content of TGFa protein varies extensively among astrocytes
and within different portions of the same cell (Fig. 6).
Hybridization histochemistry. TGFa mRNA was abundant in
cells containing small, darkly stained nuclei characteristic of
astroglial cells (Fig. 3).
Discussion
Case 2
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 TGFa 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
In the present study, we took advantage of the availability
of two HH associated with precocious puberty to determine
whether they contain cells able to produce TGFa, 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, TGFa
was abundantly expressed in astroglial cells. Importantly,
examination of one of the tumors revealed that the astrocytes
also contain EGFR, the receptor that initiates TGFa biological
actions. In both rodents and nonhuman primates, TGFa is
expressed in astroglial cells of hypothalamic regions in-
HH AND GROWTH FACTORS
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FIG. 3. Detection of TGFa mRNA in
the HH from patient 1 using a 35S-UTPlabeled human TGFa cRNA. Arrows
highlight cells showing intense hybridization. Bar, 25 mm.
FIG. 4. A, Absence of LHRH neurons in the HH from patient 2 as assessed by immunohistofluorescence-confocal microscopy. B, Detection of
LHRH neurons (red color) in the hypothalamus of an adult female rhesus monkey, using the same monoclonal antibody that failed to reveal
the presence of these cells in the HH depicted in A. C, Detection of LHRH nerve fibers (red color) in the hypothalamus of the same monkey
depicted in B. Cell nuclei are seen in blue. Bars, 20 mm.
volved in the control of gonadotropin secretion (14, 27). In
both species, hypothalamic TGFa 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 TGFa availability to pertinent regions of the neuroendocrine brain have demonstrated the
importance of local changes in TGFa 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 TGFa
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,
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FIG. 5. A, Astrocytes in the HH from patient 2, identified by immunohistofluorescence-confocal microscopy detection of GFAP. B, TGFa in cells
from the same HH. C, Colocalization of TGFa and GFAP in the same cells, visualized after merging the GFAP and TGFa images depicted in
A and B. Bars, 10 mm.
FIG. 6. Higher magnification confocal
image demonstrating the extensive colocalization of TGFa and GFAP in astroglial cells of the HH from patient 2.
Extensive colocalization is visualized as
a yellow color. Cellular areas containing
mostly GFAP are shown as a reddish
color. Cellular areas containing an
abundance of TGFa are depicted as a
greenish color.
indicating that focal increases in TGFa production must occur near LHRH neurons to accelerate the pace of female
sexual maturation.
The abundance of TGFa in astroglial cells of the two HH
studied was striking. Interestingly, most, if not all, astrocytes in the cerebellar astrocytomas examined displayed
abundant TGFa 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 TGFa content, neither cerebellar tumor was associated with precocious puberty, reinforcing the concept (16)
HH AND GROWTH FACTORS
that only a close proximity of TGFa-producing cells to the
LHRH neuronal network allows the TGFa-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 TGFa, 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 TGFa
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
TGFa would be expected to act on LHRH neurons located in
the adjacent, normal hypothalamic tissue. In addition, the
tumor may secrete TGFa 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 smallsize 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 TGFa, 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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