Activation of erbB-1 receptors by glial TGFalpha has been shown to be a component of the developmental program by which the neuroendocrine brain controls mammalian sexual development. The participation of other members of the erbB family may be required, however, for full signaling capacity. Here, we show that activation of astrocytic erbB-2/erbB-4 receptors plays a significant role in the process by which the hypothalamus controls the advent of mammalian sexual maturation. Hypothalamic astrocytes express both the erbB-2 and erbB-4 genes, but no erbB-3, and respond to neuregulins (NRGs) by releasing prostaglandin E(2) (PGE(2)), which acts on neurosecretory neurons to stimulate secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development. The actions of TGFalpha and NRGs in glia are synergistic and involve recruitment of erbB-2 as a coreceptor, via erbB-1 and erbB-4, respectively. Hypothalamic expression of both erbB-2 and erbB-4 increases first in a gonad-independent manner before the onset of puberty, and then, at the time of puberty, in a sex steroid-dependent manner. Disruption of erbB-2 synthesis in hypothalamic astrocytes by treatment with an antisense oligodeoxynucleotide inhibited the astrocytic response to NRGs and, to a lesser extent, that to TGFalpha and blocked the erbB-dependent, glia-mediated, stimulation of LHRH release. Intracerebral administration of the oligodeoxynucleotide to developing animals delayed the initiation of puberty. Thus, activation of the erbB-2-erbB-4 receptor complex appears to be a critical component of the signaling process by which astrocytes facilitate the acquisition of female reproductive capacity in mammals.

Little is known about the presence of trophic factors in the hypothalamus and the role they may play in regulating the functional development of hypothalamic neurons. We have investigated the ability of epidermal growth factor (EGF) and transforming growth factor a (TGF-a) to affect the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls reproductive development. We have also determined whether the genes encoding EGF and TGF-a are expressed in the prepubertal female hypothalamus. Northern blot analysis of poly(A)+ RNA utilizing a single-stranded EGF cDNA probe failed to reveal the presence of EGF mRNA in either the hypothalamus or the cerebral cortex at any age studied (fetal day 18 to postnatal day 36). In contrast, both a complementary RNA probe and a double-stranded TGF-a cDNA recognized in these regions a 4.5-kilobase (kb) mRNA species identical to TGF-a mRNA. The abundance of TGF-a mRNA was 3-4 times greater in the hypothalamus than in the cerebral cortex. Both EGF and TGF-a (2-100 ng/ml) elicited a dose-related increase in LHRH release from the median eminence ofjuvenile rats in vitro. They also enhanced prostaglandin E2 (PGE2) release. The transforming growth factors TGF-fi, and -P2 were ineffective. Only a high dose of basic fibroblast growth factor was able to increase LHRH and PGE2 release. Blockade of the EGF receptor transduction mechanism with RG 50864, a selective inhibitor of EGF receptor tyrosine kinase activity, prevented the effect of both EGF and TGF-a on LHRH and PGE2 release but failed to inhibit the stimulatory effect of PGE2 on LHRH release. Inhibition of prostaglandin synthesis abolished the effect of TGF-et on LHRH, indicating that PGE2 mediates TGF-ainduced LHRH release. The results indicate that the effect of EGF and TGF-a on LHRH release is mediated by the EGF/TGF-a receptor and suggest that TGF-a rather than EGF may be the physiological ligand for this interaction. Since in the central nervous system most EGF/TGF-a receptors are located on glial cells, the results also raise the possibility that-at the median eminence-TGF-a action may involve a glial-neuronal interaction, a mechanism by which the trophic factor first stimulates PGE2 release from glial cells, and then PGE2 elicits LHRH from the neuronal terminals.

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Studies in female rats have shown that transforming growth factor alpha (TGFα) stimulates release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation, and that expression of the TGFα gene in the hypothalamus increases during both the initiation of normal puberty and after hypothalamic lesions that induce sexual precocity. Since blockade of epidermal growth factor receptors (EGFR), which mediate TGFα actions, delayed the normal timing of puberty, it was postulated that TGFα/EGFR contributes to the neuroendocrine process that underlies the initiation of normal female puberty. The present study was undertaken to examine the hypothesis that hypothalamic expression of the TGFα gene and its receptor changes in relation to the stage of sexual development in nonhuman primates, and to determine whether these changes are accompanied by corresponding alterations in LHRH gene expression. DNA fragments complementary to the coding regions of the rhesus monkey TGFα, EGFR and LHRH genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR), sequenced and used to prepare monkey-specific antisense RNA probes. A quantitative RT-PCR was developed in which the cloned sequences were utilized to prepare RNA standards for the quantitation of tissue mRNA levels. Both TGFα and EGFR mRNA levels in the medial basal hypothalamus and preoptic area of female monkeys were elevated during neonatal life (1 week to 6 months of age), when FSH secretion is also high, decreased during juvenile development (8–18 months of age) when secretion of both FSH and LH is low, and markedly increased during the expected time of puberty (30-36 months of age). No such changes were observed in either the cerebellum or the cerebral cortex, two brain regions irrelevant to neuroendocrine reproductive control. In contrast to the pronounced alterations in hypothalamic TGFα/EGFR gene expression observed during sexual development, LHRH mRNA levels did not vary significantly during this time. Hybridization histochemistry revealed the presence of both TGFα and EGFR mRNAs in cells scattered throughout the hypothalamus, but more predominantly in the median eminence, suprachiasmatic nuclei, optic chiasm and cells along the wall of the third ventricle. These results demonstrate that increases in TGFα and EGFR gene expression, specific to the neuroendocrine brain, occur during developmental phases in which gonadotropin output is also elevated – most noticeably at the time of puberty. The remarkable parallelism between these changes and those in gonadotropin secretion known to occur during postnatal sexual development in primates, and the lack of developmental alterations in LHRH mRNA levels suggest that, as postulated in rodents, an activation of the TGFα ligand-EGF receptor system leading to LHRH gene-independent changes in LHRH release contributes to the the neuroendocrine control of female puberty in primates.