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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.

Prostaglandin E2 (PGE2) mediates the stimulatory effect of norepinephrine (NE) on the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling reproductive function. In rodents, this facilitatory effect requires previous exposure to estradiol, suggesting that the steroid affects downstream components in the cascade that leads to PGE2-induced LHRH release. Because astroglia are the predominant cell type contacting LHRH-secreting nerve terminals, we investigated the involvement of hypothalamic astrocytes in the estradiol facilitation of PGE2-induced LHRH release. A subpopulation of LHRH neurons was found to express the mRNA encoding the PGE2 receptor subtype EP1-R, which is coupled to calcium mobilization. The LHRH-producing cell line GT1-1 also contains EP1-R mRNA and, to a lesser extent, the three alternatively spliced forms of EP3-R mRNA (alpha, beta, and gamma) that encode receptors linked to inhibition and stimulation of cAMP formation. Hypothalamic astrocytes treated with estradiol produced a conditioned medium that when applied to GT1-1 cells resulted in a selective upregulation of EP1-R and EP3gamma-R mRNAs. The conditioned medium also enhanced the LHRH response to EP1-R and EP3-R agonists and the cAMP response to EP3-R activation. Thus, one mechanism by which estradiol facilitates the effect of neurotransmitters acting via PGE2 to stimulate LHRH release is by enhancing the glial production of substances that upregulate PGE2 receptors on LHRH neurons. The existence of such a mechanism underscores the emerging importance of glial-neuronal communication in the control of brain neurosecretory activity.

Previous studies demonstrated the involvement of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, in the developmental regulation of hypothalamic LHRH release. Although both TGF alpha and EGF stimulate LHRH release, they do not appear to act directly on LHRH neurons, as no EGF/TGF alpha receptors are detected on these cells in vivo. Instead, the stimulatory effect of TGF alpha on LHRH release seems to require a glial intermediacy. The present study identifies one of the glial molecules involved in this process. In vitro exposure of purified hypothalamic astrocytes to TGF alpha or EGF in a defined medium led to activation of the cyclooxygenase-mediated pathway of arachidonic acid metabolism, as indicated by an increase in PGE2 release, but failed to affect lipooxygenase-mediated metabolism, as assessed by the lack of increase in leukotriene C4 production; addition of TGF alpha- (T-CM) or EGF-conditioned medium to cultures of LHRH-producing GT1-1 cells stimulated LHRH release. In contrast, direct exposure of GT1-1 cells to the growth factors was ineffective. Incubation of the cells in medium conditioned by untreated astrocytes (CM) was also ineffective. Blockade of either EGF receptor signal transduction or cyclooxygenase activity in the astrocytic cultures prevented both TGF alpha-induced PGE2 formation in astrocytes and the stimulatory effect of T-CM on LHRH release. Immunoneutralization of PGE2 actions or selective removal of the PG from T-CM also prevented T-CM-induced LHRH release. Addition of exogenous PGE2 restored the effect. Thus, PGE2 is one of the glial molecules involved in mediating the stimulatory effect of TGF alpha on LHRH release. The effectiveness of PGE2 in eliciting LHRH release was, however, greatly reduced when PG was delivered to GT1-1 cells in astrocyte-defined medium instead of CM. Thus, astrocytes appear to produce a yet to be identified substance(s) that facilitates the stimulatory effect of PGE2 on LHRH output. We postulate that the ability of TGF alpha to enhance LHRH release depends on the potentiating interaction of PGE2 with these additional glial-derived molecules.

Studies in female rats have shown that transforming growth factor alpha (TGF alpha) stimulates release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual maturation, and that expression of the TGF alpha 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 TGF alpha actions, delayed the normal timing of puberty, it was postulated that TGF alpha/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 TGF alpha 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 TGF alpha, 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 TGF alpha 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 TGF alpha/EGFR gene expression observed during sexual development, LHRH mRNA levels did not vary significantly during this time. Hybridization histochemistry revealed the presence of both TGF alpha 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 TGF alpha 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent findings have led to the concept that transforming growth factor alpha (TGF alpha) contributes to the neuroendocrine regulation of female puberty by stimulating the release of luteinizing hormone-releasing hormone (LHRH), the neurohormone controlling sexual development. It was postulated that this effect is mediated by epidermal growth factor receptors (EGFR) and that EGFR may not be located on LHRH neurons, so that TGF alpha-induced LHRH release would require an intermediate cell-to-cell interaction, presumably of glial-neuronal nature. The present study was undertaken to characterize the presence of EGFR in rat hypothalamus and to determine if changes in EGFR gene expression and EGFR protein occur at the time of puberty. RNA blot hybridization demonstrated that the hypothalamus expresses all mRNA species known to encode EGFR. RNase protection assays revealed that alternative splicing of the EGFR primary mRNA transcript occurs in the hypothalamus and produces a predominant transcript encoding the full-length EGFR and a much less abundant, shorter mRNA encoding a truncated, and presumably secreted form of EGFR. EGFR-like immunoreactive material was found in several hypothalamic regions including the organum vasculosum of the lamina terminalis, supraoptic, suprachiasmatic, and paraventricular nuclei, ependymal cells lining the third ventricle, some astrocytes associated with blood vessels, astrocytes of the pial surface, and tanycytes and glial cells of the median eminence (ME). Low levels of EGFR mRNA were detected by hybridization histochemistry in cells of the same areas containing EGFR-like immunoreactivity. Double-immunohistochemistry revealed that even though LHRH neurons are in close proximity to EGFR-positive cells, they do not contain EGFR. In the ME, EGFR-immunonegative LHRH nerve terminals tightly coexist with EGFR-positive cells, presumably tanycytes and glial astrocytes. EGFR mRNA levels measured by quantitative reverse transcription-polymerase chain reaction assay (RT-PCR) in the ME-arcuate nucleus region at the time of puberty decreased in the morning of the first proestrus, i.e., preceding the first preovulatory surge of gonadotropins, and rebounded at the time of the surge. Functional EGFR protein levels, detected by the ability of the receptor to autophosphorylate in response to ligand or divalent antibody-induced activation, changed in a similar manner at the time of puberty. No such changes were observed in the cerebellum, a brain region irrelevant to neuroendocrine reproductive control. These results demonstrate the existence of EGF receptors in the prepubertal female rat hypothalamus and suggest that changes in EGFR gene expression and biologically active EGFR protein contributes to the neuroendocrine process underlying the first preovulatory surge of gonadotropins.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypothalamic injury causes female sexual precocity by activating luteinizing hormone-releasing hormone (LHRH) neurons, which control sexual development. Transforming growth factor-alpha (TGF-alpha) has been implicated in this process, but its involvement in normal sexual maturation is unknown. The present study addresses this issue. TGF-alpha mRNA and protein were found mostly in astroglia, in regions of the hypothalamus concerned with LHRH control. Hypothalamic TGF-alpha mRNA levels increased at times when secretion of pituitary gonadotropins--an LHRH-dependent event--was elevated, particularly at the time of puberty. Gonadal steroids involved in the control of LHRH secretion increased TGF-alpha mRNA levels. Blockade of TGF-alpha action in the median eminence, a site of glial-LHRH nerve terminal association, delayed puberty. These results suggest that TGF-alpha of glial origin is a component of the developmental program by which the brain controls mammalian sexual maturation.

It has long been known that lesions of the hypothalamus lead to female sexual precocity. While an increased production of luteinizing hormone-releasing hormone (LHRH), the neurohormone that controls sexual development, appears to mediate the advancement of puberty induced by these lesions, little is known about the mechanism(s) by which hypothalamic injury activates LHRH secretion. Since brain lesions result in accumulation of neurotrophic/mitogenic activities in the injured area, we tested the hypothesis that transforming growth factor alpha (TGF-alpha), a mitogenic polypeptide recently shown to stimulate LHRH release, is produced in response to hypothalamic injury and mediates the effect of the lesion on puberty. Radiofrequency lesions of the preoptic area-anterior hypothalamic area (POA-AHA) of 22-day-old female rats resulted in precocious puberty within 7 days after the operation. RNA blot hybridization revealed that lesion-induced puberty was preceded by an increase in TGF-alpha mRNA levels in the POA-AHA. Epidermal growth factor (EGF) mRNA was undetectable in both intact and lesioned hypothalami. TGF-alpha mRNA levels, quantitated by RNase protection assays, were 3.5-fold greater in lesioned animals approaching puberty than in age-matched controls. Immunohistochemical studies, utilizing single- and double-staining procedures, demonstrated the presence of TGF-alpha precursor-like immunoreactivity in reactive astrocytes surrounding the lesion site. Hybridization histochemistry showed increased TGF-alpha mRNA expression in cells of the same area, further implicating reactive astrocytes as a site of TGF-alpha synthesis. The actions of TGF-alpha are mediated by its interaction with EGF receptors. Continuous infusion of RG-50864, an inhibitor of EGF receptor kinase activity, at the site of injury prevented the advancement of puberty induced by the lesion. These results suggest that TGF-alpha acting via EGF-like receptors contributes to the acceleration of puberty induced by anterior hypothalamic lesions. They also indicate that activation of TGF-alpha gene expression in glial cells is a component of the hypothalamic response to injury.

The content of GnRH and its precursor peptide were quantified in female rats bearing lesions in the anterior medial preoptic nucleus (AMPO) and the suprachiasmatic nucleus (SCN), and the effects of the lesions on the synthetic activity of the GnRH neurons were evaluated. Electrolytic lesions which induced persistent estrous (PE), or irregular estrous cycles, were produced by passing 5-10 microA of direct current into the AMPO or the SCN of female rats which exhibited regular 4 days estrous cycles before the lesions. Approximately 5 weeks after lesion placement, blood samples were withdrawn from catheterized, freely moving animals and plasma LH, PRL, estrogen, and progesterone were determined by RIA. The preovulatory surges of LH and PRL were eliminated in AMPO- or SCN-lesioned PE rats. Moreover, the LH surge was eliminated and the PRL surge significantly attenuated after estrogen and progesterone treatment of rats bearing complete lesions, irrespective of the presence of ovaries. Irregular cycling animals with incomplete AMPO or SCN lesions, exhibited attenuated LH surge and PRL surge similar to proestrous controls. In one incidence this occurred spontaneously, and could also be induced by sequential estrogen and progesterone injections. After ovariectomy, plasma LH levels were significantly lower in the lesioned animals as compared to sham operated rats (P less than 0.05). Similar secretory patterns of LH and PRL were obtained from a second series of sham-operated rats during the different stages of the estrous cycle or from AMPO- or SCN-lesioned rats during persistent estrus. After 2 months the animals were killed between 0830 and 0930 h, and the preoptic area and the basal hypothalamus were microdissected from the brain sections. After extraction and purification, proGnRH and GnRH levels were measured by RIA. ProGnRH levels in the preoptic area were significantly reduced in AMPO- or in SCN-lesioned rats, compared to proestrous controls (P less than 0.01). In contrast, GnRH levels in either area did not differ in AMPO- or in SCN-lesioned animals compared to sham-operated, proestrous controls. Therefore, lesions of the AMPO or the SCN produce PE and reduce proGnRH without reducing GnRH levels. These data would suggest that the AMPO and the SCN participate in the control of the estrous cycle and are necessary for preovulatory surges of PRL and LH to occur and that the AMPO and the SCN form part of the neural circuit that regulates GnRH synthesis and/or release.