Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
0021-972X/99/$03.00/0 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- 4695 4696 JCE & M • 1999 Vol 84 • No 12 JUNG ET AL. 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. 4697 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), 4698 JCE & M • 1999 Vol 84 • No 12 JUNG ET AL. 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 4699 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, 4700 JUNG ET AL. JCE & M • 1999 Vol 84 • No 12 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. References 1. Lichtenstein BW. 1971 Hamartomas and phacomatoses. In: Minckler J, ed. Pathology of the Nervous System, Vol 2. New York: McGraw-Hill; 1897–1906. 2. Zuniga OF, Tanner SM, Wild WO, et al. 1983 Hamartoma of CNS associated with precocious puberty. Am J Dis Child. 137:127–133. 3. Driggs M, Spatz H. 1939 Pubertas praecox bei einer hyperplastischen Mibbildung des Tuber cinereum. Virchaus Archiv Pathol Anat. 305:567–592. 4. Richter RB. 1951 True hamartoma of the hypothalamus associated with pubertas praecox. J Neuropathol Exp Neurol. 4:368 –383. 5. Starceski PJ, Lee PA, Albright AL, et al. 1990 Hypothalamic hamartomas and sexual precocity. Am J Dis Child. 144:225–228. 6. Berkovic SF, Kuzniecky RI, Andermann F. 1997 Human epileptogenesis and hypothalamic hamartomas: new lessons from an experiment of nature. Epilepsia. 38:1–3. 7. Valdueza JM, Cristante L, Dammann O, et al. 1994 Hypothalamic hamartomas: with special reference to gelastic epilepsy and surgery. Neurosurgery. 34:949 –958. 8. Grumbach MM, Styne DM. 1992 Puberty: ontogeny, neuroendocrinology, physiology, and disorders. In: Wilson JD, Foster DW, eds. Williams Textbook of Endocrinology, 8th Ed. Philadelphia: W.B. Saunders, Co.; 1139 –1221. 9. Judge DM, Kulin HE, Page R, et al. 1977 Hypothalamic hamartoma: a source 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 4701 of luteinizing-hormone-releasing factor in precocious puberty. N Engl J Med. 296:7–10. Culler FL, James HE, Simon ML, et al. 1985 Identification of gonadotropinreleasing hormone in neurons of a hypothalamic hamartoma in a boy with precocious puberty. Neurosurgery. 17:408 – 412. Price RA, Lee PA, Albright AL, et al. 1984 Treatment of sexual precocity by removal of a luteinizing hormone-releasing hormone secreting hamartoma. JAMA. 251:2247–2249. Hochman HI, Judge DM, Reichlin S. 1981 Precocious puberty and hypothalamic hamartoma. Pediatrics. 67:236 –244. Ojeda SR. 1994 The neurobiology of mammalian puberty: has the contribution of glial cells been underestimated? J NIH Res. 6:51–56. Ma YJ, Junier M-P, Costa ME, et al. 1992 Transforming growth factor a (TGFa) gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation. Neuron. 9:657– 670. Junier M-P, Ma YJ, Costa ME, et al. 1991 Transforming growth factor a contributes to the mechanism by which hypothalamic injury induces precocious puberty. Proc Natl Acad Sci USA. 88:9743–9747. Rage F, Hill DF, Sena-Esteves M, et al. 1997 Targeting transforming growth factor a expression to discrete loci of the neuroendocrine brain induces female sexual precocity. Proc Natl Acad Sci USA. 94:2735–2740. Mahachoklertwattana P, Kaplan SL, Grumbach MM. 1993 The luteinizing hormone-releasing hormone-secreting hypothalamic hamartoma is a congenital malformation: natural history. Clin Endocrinol Metab. 77:118 –124. Partsch CJ, Hummelink R, Peter M, et al. 1993 Comparison of complete and incomplete suppression of pituitary-gonadal activity in girls with central precocious puberty: influence on growth and predicted final height. The GermanDutch Precocious Puberty Study Group. Horm Res. 39:111–117. Urbanski HF. 1991 Monoclonal antibodies to luteinizing hormone-releasing hormone: production, characterization, and immunocytochemical application. Biol Reprod. 44:681– 686. Ronnekleiv OK, Adelman JP, Weber E, et al. 1987 Immunohistochemical demonstration of proGnRH and GnRH preoptic-basal hypothalamus of the primate. Neuroendocrinology. 45:518 –521. Gentry LE, Twardzik DR, Lim GJ, et al. 1987 Expression and characterization of transforming growth factor a precursor protein in transfected mammalian cells. Mol Cell Biol. 7:1585–1591. Kris RM, Lax I, Gullick W, et al. 1985 Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein. Cell. 40:619 – 625. Lakos S, Basbaum AI. 1986 Benzidine dihydrochloride as a chromogen for single- and double-label light and electron microscopic immunocytochemical studies. J Histochem Cytochem. 34:1047–1056. Ma YJ, Hill DF, Junier M-P, et al. 1994 Expression of epidermal growth factor receptor changes in the hypothalamus during the onset of female puberty. Mol Cell Neurosci. 5:246 –262. Simmons DM, Arriza JL, Swanson LW. 1989 A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radiolabeled single-stranded RNA probes. J Histotechnol. 12:169 –181. Jhappan C, Stahle C, Harkins RN, et al. 1990 TGFa overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell. 61:1137–1146. Ma YJ, Costa ME, Ojeda SR. 1994 Developmental expression of the genes encoding transforming growth factor a (TGFa) and its receptor in the hypothalamus of female rhesus macaques. Neuroendocrinology. 60:346 –359. Junier M-P, Hill DF, Costa ME, et al. 1993 Hypothalamic lesions that induce female precocious puberty activate glial expression of the epidermal growth factor receptor gene: differential regulation of alternatively spliced transcripts. J Neurosci. 13:703–713. Kiss R, Dewitte O, Decaestecker C, et al. 1997 The combined determination of proliferative activity and cell density in the prognosis of adult patients with supratentorial high-grade astrocytic tumors. Am J Clin Pathol. 107:321–331. Reubi JC, Horisberger U, Lang W, et al. 1989 Coincidence of EGF receptors and somatostatin receptors in meningiomas but inverse, differentiationdependent relationship in glial tumors. Am J Pathol. 134:337–344. Markin RS, Leibrock LG, Huseman CA, et al. 1987 Hypothalamic hamartoma: a report of 2 cases. Pediatr Neurosci. 13:19 –26. Kammer KS, Perlman K, Humphreys RP, et al. 1980 Clinical and surgical aspects of hypothalamic hamartoma associated with precocious puberty in a 15-month-old boy. Child’s Brain. 6:150 –157. Ma YJ, Berg-von der Emde K, Rage F, et al. 1997 Hypothalamic astrocytes respond to transforming growth factor a with secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone. Endocrinology. 138:19 –25. McCullagh EP, Rosenberg HS, Norman N. 1960 Tumor of the tuber cinereum with precocious puberty: case report with hormone assays. J Clin Endocrinol Metab. 20:1286 –1293.