Search

It is becoming increasingly clear that astroglial cells are active participants in the process by which information is generated and disseminated within the central nervous system (CNS). In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons. They contribute to facilitating sexual development by stimulating the release of luteinizing hormone-releasing hormone (LHRH), the neuropeptide that controls sexual development, from LHRH neurons. Astrocytes secrete several growth factors able to stimulate LHRH secretion. Two members of the epidermal growth factor (EGF) family--transforming growth factor alpha (TGFalpha) and the neuregulins (NRGs)-are produced in hypothalamic astrocytes and elicit LHRH secretion indirectly, via activation of receptor complexes formed by three members of the EGF receptor family, also located on astrocytes. Activation of these receptors results in the production of at least one neuroactive substance, prostaglandin E2 (PGE2), which stimulates LHRH secretion upon binding to specific receptors on LHRH neurons. Overexpression of TGFalpha in the hypothalamus accelerates puberty, whereas blockade of either TGFalpha or NRG actions delays the process, indicating that both peptides are physiological components of the neuroendocrine mechanism that controls sexual maturation. An increase in hypothalamic expression of at least two of the erbB receptors is initiated before the pubertal augmentation of gonadal steroid secretion and is completed on the day of the first preovulatory surge of gonadotropins. This secondary increase is brought about by gonadal steroids. Estrogen and progesterone facilitate erbB-mediated glia-to-LHRH neuron communication by enhancing astrocytic gene expression of at least one of the EGF-related ligands (TGFalpha) and two of the receptors (erbB-2 and erbB-4). They also facilitate the LHRH response to PGE2 via induction of PGE2 receptors in LHRH neurons. A search for genes that may act as upstream regulators of the pubertal process resulted in the identification of two potential candidates: Oct-2, a POU domain gene originally described in cells of the immune system, and TTF-1, a member of the Nkx family of homeodomain transcriptional regulators required for diencephalic morphogenesis. The hypothalamic expression of both genes increases during juvenile development before the first hormonal manifestations of puberty take place. Their mRNA transcripts are localized to specific hypothalamic cellular subsets, where they appear to regulate different, but interactive, components of the neuronal-glial complex controlling LHRH secretion. While Oct-2 transactivates the TGFalpha promoter, TTF-1 does so to the erbB-2 and LHRH genes but inhibits preproenkephalin promoter activity, suggesting that both transcriptional regulators may act coordinately in the normal hypothalamus to activate genes involved in facilitating the advent of puberty and repress those restraining sexual development. Altogether, these observations indicate that the central activation of the pubertal process involves the participation of both neuronal and astroglial networks and the contribution of upstream transcriptional regulators acting on both the neuronal and glial components of the system.

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.

It is now clear that astroglial cells actively contribute to both the generation and flow of information within the central nervous system. In the hypothalamus, astrocytes regulate the secretory activity of neuroendocrine neurons. A small subset of these neurons secrete luteinizing hormone-releasing hormone (LHRH), a neuropeptide essential for sexual development and adult reproductive function. Astrocytes stimulate LHRH secretion via cell-cell signaling mechanisms involving growth factors recognized by receptors with either serine/threonine or tyrosine kinase activity. Two members of the epidermal growth factor (EGF) family and their respective tyrosine kinase receptors appear to play key roles in this regulatory process. Transforming growth factor-alpha (TGFalpha) and its distant congeners, the neuregulins (NRGs), are produced in hypothalamic astrocytes. They stimulate LHRH secretion indirectly, via activation of erbB-1/erbB-2 and erbB-4/erbB-2 receptor complexes also located on astrocytes. Activation of these receptors leads to release of prostaglandin E(2) (PGE(2)), which then binds to specific receptors on LHRH neurons to elicit LHRH secretion. Gonadal steroids facilitate this glia-to-neuron communication process by acting at three different steps along the signaling pathway. They (a) increase astrocytic gene expression of at least one of the EGF-related ligands (TGFalpha), (b) increase expression of at least two of the receptors (erbB-4 and erbB-2), and (c) enhance the LHRH response to PGE(2) by up-regulating in LHRH neurons the expression of specific PGE(2) receptor isoforms. Focal overexpression of TGFalpha in either the median eminence or preoptic area of the hypothalamus accelerates puberty. Conversely, blockade of either TGFalpha or NRG hypothalamic actions delays the process. Thus, both TGFalpha and NRGs appear to be physiological components of the central neuroendocrine mechanism controlling the initiation of female puberty. By facilitating growth factor signaling pathways in the hypothalamus, ovarian steroids accelerate the pace and progression of the pubertal process., (Copyright 1999 John Wiley & Sons, Inc.)

POU homeodomain genes are transcriptional regulators that control development of the mammalian forebrain. Although they are mostly active during embryonic life, some of them remain expressed in the postnatal hypothalamus, suggesting their involvement in regulating differentiated functions of the neuroendocrine brain. We show here that Oct-2, a POU domain gene originally described in cells of the immune system, is one of the controlling components of the cell-cell signaling process underlying the hypothalamic regulation of female puberty. Lesions of the anterior hypothalamus cause sexual precocity and recapitulate some of the events leading to the normal initiation of puberty. Prominent among these events is an increased astrocytic expression of the gene encoding transforming growth factor-alpha (TGF alpha), a tropic polypeptide involved in the stimulatory control of LHRH secretion. The present study shows that such lesions result in the rapid and selective increase in Oct-2 transcripts in TGF alpha-containing astrocytes surrounding the lesion site. In both lesion-induced and normal puberty, there is a preferential increase in hypothalamic expression of the Oct-2a and Oct-2c alternatively spliced messenger RNA forms of the Oct-2 gene, with an increase in 2a messenger RNA levels preceding that in 2c and antedating the peripubertal activation of gonadal steroid secretion. Both Oct-2a and 2c trans-activate the TGF alpha gene via recognition motifs contained in the TGF alpha gene promoter. Inhibition of Oct-2 synthesis reduces TGF alpha expression in astroglial cells and delays the initiation of puberty. These results suggest that the Oct-2 gene is one of the upstream components of the glia to neuron signaling process that controls the onset of female puberty in mammals.

In recent years evidence has begun to accumulate indicating that the central control of mammalian puberty requires not only changes in transsynaptic communication, but also the participation of glial cells. Neurons and astrocytes control the pubertal process by regulating the secretory activity of those neurons that produce luteinizing hormone-releasing hormone (LHRH), the neuropeptide that governs sexual development. LHRH, in turn, directs sexual development by stimulating the secretion of pituitary gonadotropins. Astrocytes affect LHRH neuronal function via cell-cell signaling mechanisms involving several growth factors acting via receptors endowed with tyrosine kinase activity. We have identified two members of the epidermal growth factor/transforming growth factor alpha (EGF/TGFalpha) family and their respective receptors as key players in the glial-neuronal interactive process that regulates LHRH secretion. Our results indicate that TGFalpha and its distant congener neuregulin (NRG) are produced in hypothalamic astrocytes and stimulate LHRH release indirectly via activation of their respective receptors, located--surprisingly--not on LHRH neurons, but on astrocytes. Activation of EGF receptors by TGFalpha, and/or the erbB2/erbB4 receptor complex by NRG, leads to glial release of prostaglandin (PG) E2, which then acts directly on LHRH neurons to stimulate LHRH release. That a central blockade of TGFalpha or NRG action delays puberty, and focal overexpression of TGFalpha advances it, leads to the conclusion that both TGFalpha and NRG are physiological components of the central mechanism controlling the initiation of female puberty.

Emerging evidence suggests that, in addition to neuronal inputs, growth factors of glial origin are also important in the control of mammalian puberty via a cell-cell interaction that ultimately affects the neurons that release gonadotropin-releasing hormone (GnRH), a neurohormone controlling sexual development. Among these growth factors, transforming growth factor-alpha (TGF alpha) appears to be one of the physiologic components that controls the onset of female puberty by affecting GnRH neuronal activity in a glia-mediated autocrine/paracrine manner. Specifically, TGF alpha induces glia to produce bioactive substances, such as prostaglandin E2 (PGE2). In turn, PGE2 directly acts on GnRH neurons to stimulate the release of GnRH. Furthermore, the neuroregulin of glial origin neu differentiation factor (NDF) was found to facilitate the action of TGF alpha, suggesting that other growth factors may exert their biologic effects on GnRH neuronal function via a glia/neuron interaction. Another indication that glial cells may be involved in the regulation of neuroendocrine function is the presence of estrogen receptors on hypothalamic astrocytes. Thus, region-specific glial cells appear to play an integral role in the regulation of neuroendocrine function.

The importance of transforming growth factor-alpha (TGF alpha) in female reproductive development was assessed using transgenic mice bearing a human TGF alpha complementary DNA under the control of a mouse metallothionein-1 promoter (MT1-hTGF alpha). Examination of the brain and ovaries 5 h after a single sc injection of zinc chloride, administered to activate the MT1-hTGF alpha transgene, revealed that prominent sites of human TGF alpha messenger RNA expression within these tissues were the hypothalamus and ovarian follicles, respectively. In vitro experiments showed that acute transgene activation increased hypothalamic release of LH-releasing hormone. In contrast, the ovarian steroidal response to gonadotropins, examined in vitro, was markedly attenuated. Chronic activation of transgene expression by daily administration of zinc chloride delayed the time of first estrus (an index of peripubertal estrogen secretion), but shortened the interval between first estrus and the onset of estrous cyclicity (an index of reproductive competence). Accumulation of small antral follicles, accompanied by thecal hypertrophy and enhanced androgen production, preceded the acquisition of ovulatory capacity. These changes were accompanied by reduced serum LH levels, suggesting that the relative inability of small antral follicles to develop further in TGF alpha-overexpressing mice is at least in part due to inappropriate gonadotropin support. Serum LH levels in these animals may be reduced by an augmented androgen negative feedback signal. Nontransgenic mouse ovaries, placed under the control of a transgenic hypothalamus by heterologous grafting, rapidly ovulated and initiated estrous cyclicity. In contrast, acquisition of reproductive capacity was severely delayed in nontransgenic mice bearing transgenic ovarian grafts. The results indicate that TGF alpha regulates female reproductive development through two opposing mechanisms: within the brain, it facilitates the neuroendocrine activation of the process; at the ovarian level, modulates the stimulatory effect of gonadotropin hormones on follicular growth and steroidogenesis.

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.

Recent evidence has implicated the transforming growth factor-alpha (TGF alpha)/epidermal growth factor receptor (EGFR) system in the mechanism by which hypothalamic lesions accelerate female sexual development. Since acquisition and maintenance of reproductive functions depend on the secretory activity of LHRH neurons, the present studies were undertaken to characterize some of the cellular and molecular events that underlie lesion-induced activation of the LHRH neuronal network. Bilateral electrolytic lesions of the posterior portion of the preoptic region and anterior hypothalamic area (POA-AHA) in 22-day-old rats resulted in vaginal opening and ovulation within 7 days. Morphological maturation of LHRH neurons was assessed by the relative frequency of irregular and smooth neurons (the former being the predominant type in adult animals). Within 20 h after the lesion, there was a significant decrease in the proportion of LHRH neurons with spiny irregular contours, indicating reversal to a more immature morphological type. This change was followed by accelerated spine reformation, so that at the time of precocious proestrus, the incidence of irregular LHRH neurons was similar in lesioned and age-matched control rats. A striking increase in c-fos mRNA levels occurred within 1 h after the lesion in the area neighboring the site of injury, reflecting the immediate cell response to trauma. Immunohistochemical localization of the c-fos protein, used to estimate changes in cellular activity at the single cell level, demonstrated c-fos induction in unidentified cells near the lesion and astrocytes, but not in LHRH neurons 20 h after injury. In contrast, a selective increase in c-fos expression was observed in LHRH neurons during the initiation of precocious puberty 5-7 days later at the time of the first proestrus. An increase in plasma LH associated with a drop in LHRH content in the median eminence and an increase in pro-LHRH precursor in the POA-AHA, with no changes in LHRH mRNA, was found to antedate the first preovulatory surge of gonadotropins in lesioned rats. Assessment of the changes in PC2 mRNA, which encodes a novel dibasic endoprotease presumptively involved in tissue-specific processing of a class of prohormones that includes pro-LHRH, showed that the content of PC2 mRNA in the AHA-POA increases during normal puberty, but not in lesioned animals, thus providing a potential explanation for the divergent changes in pro-LHRH and mature decapeptide found in lesioned rats.(ABSTRACT TRUNCATED AT 400 WORDS)

We previously demonstrated that the immature rat ovary synthesizes nerve growth factor (NGF), and that interference of NGF actions by immunoneutralization during neonatal life prevents development of the ovarian sympathetic innervation and delays follicular maturation. Since the actions of NGF are exerted via binding to specific cell surface receptors, the present study was undertaken to define and characterize the presence of NGF receptors (NGFrec) in the developing rat ovary. NGF interacts with two classes of NGFrec. The most abundant is a low affinity form expressed in the central nervous system and peripheral tissues. This receptor is encoded by a single 3.8-kilobase mRNA species. Cross-linking of [125I]NGF to ovarian membranes followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography showed the presence of a approximately 90-kilodalton molecular species which corresponds in size to the predominant NGF receptor species cross-linked to its ligand. While ovarian NGFrec may be of neuronal origin and reach the gland exclusively by anterograde axonal transport, RNA blot hybridization demonstrated that the ovary expresses the NGFrec mRNA species that encodes the low affinity NGF receptor and, thus, implicated the ovary itself as a site of NGFrec synthesis. NGFrec mRNA levels decreased abruptly after the first ovulation, suggesting that NGFrec may be synthesized in growing follicles and that this capacity is lost after follicular rupture and luteinization.(ABSTRACT TRUNCATED AT 250 WORDS)