RESUMO
Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Several non-coat polypeptides appeared to segregate exclusively with different populations as resolved by two-dimensional electrophoresis. Stripped CVs also exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetylcholine esterase, and an Mr 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination.
Assuntos
Clatrina/metabolismo , Fígado/ultraestrutura , Organoides/ultraestrutura , Animais , Receptor de Asialoglicoproteína , Proteínas de Transporte/metabolismo , Fracionamento Celular/métodos , Invaginações Revestidas da Membrana Celular/ultraestrutura , Eletroforese em Gel de Ágar/métodos , Endocitose , Exocitose , Glicoproteínas/metabolismo , Ponto Isoelétrico , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Peso Molecular , Organoides/metabolismo , Ratos , Receptor IGF Tipo 2 , Receptores Imunológicos/metabolismoRESUMO
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.
Assuntos
Astrócitos/fisiologia , Córtex Cerebral/fisiologia , Receptores ErbB/fisiologia , Hipotálamo/fisiologia , Neurregulinas/fisiologia , Receptor ErbB-2/fisiologia , Maturidade Sexual/fisiologia , Animais , Neoplasias da Mama , Córtex Cerebral/crescimento & desenvolvimento , Ventrículos Cerebrais/efeitos dos fármacos , Ventrículos Cerebrais/fisiologia , Dinoprostona/sangue , Receptores ErbB/genética , Estradiol/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes erbB-1 , Humanos , Hipotálamo/crescimento & desenvolvimento , Neurregulinas/genética , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Oncogênicas v-erbB , Ovariectomia , Fosforilação , Fosfotirosina/metabolismo , Gravidez , Progesterona/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/genética , Receptor ErbB-4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
We have isolated two sheep IGF-II clones which have the same coding regions but have different 5'-untranslated regions. There is no homology between these 5'-UTR sequences which are homologous to exons 5 and 6 of the human IGFII gene. In humans these exons are transcribed only during fetal development, but in the sheep these transcripts were detected up to 28 weeks after parturition. The sizes of the sheep IGF-II mRNA transcripts are the same as those observed for the human gene, suggesting structural and transcriptional similarity between the human and sheep IGF-II genes. The sheep could therefore be a good model system in which to study IGF-II expression.
Assuntos
DNA/isolamento & purificação , Fator de Crescimento Insulin-Like II/genética , Animais , Éxons , Feminino , Trabalho de Parto , Modelos Biológicos , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/análise , OvinosRESUMO
The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host. Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a rho-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.
Assuntos
Bacteriófagos/genética , DNA Viral , Genes Virais , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Replicação do DNA , DNA Viral/biossíntese , Regulação Viral da Expressão Gênica , Dados de Sequência Molecular , Morfogênese/genética , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Ribossomos/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
A 32 kilobase-pair fragment of intron 7 of the human dystrophin gene has been sequenced and analysed for the presence of repetitive elements and open reading frames. Two transposon-like human elements (THE-1 sequences), and three intervening, and related, long terminal repeat elements, together with three Alu sequences and a LINE sequence have been identified. These represent an unexpected clustering of highly-repetitive sequences within this single segment of intron DNA. Amplification of a region of chimpanzee genomic DNA by the polymerase chain reaction has provided evidence that at least one of the THE-1 sequences is present in the same position in the chimpanzee genome and the high homology between the human and chimpanzee sequences indicates that this element was fixed within the ancestral genome before the divergence of the two species. The possible role of repetitive, transposon-like sequences in natural mutagenesis of the dystrophin gene is discussed.
Assuntos
Elementos de DNA Transponíveis , Distrofina/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Genes , Humanos , Íntrons , Dados de Sequência Molecular , Translocação Genética , Cromossomo XRESUMO
Three male F1 hybrids between Père David's deer and red deer were mated to red deer to produce 143 backcross calves. The pedigrees are a rare example of a fertile hybrid between evolutionarily divergent species. We examined the use of these families for genetic mapping of evolutionarily conserved (Type I) loci by testing for genetic linkage between five species-specific protein variants and 12 conserved DNA probes. Two probes were homologous, and the remainder syntenic, to the protein coding loci in cattle or humans. Using six restriction enzymes, each DNA probe detected one or more restriction fragments specific to Père David's deer. Linkage analyses among the species-specific variants placed the loci into four linkage groups within which linkage between adjacent loci and gene order was supported by a LOD > 3. The linkage groups were (HPX, HBB)-FSHB-ACP2, LDHA-CD5-IGF2, BMP3-(GC, ALB)-(KIT, PDGFRA) and LDLR-C3-FGF1. Southern and protein analysis of LDHA and ALB provided identical segregation data. These linkage groups were consistent with the cattle gene map and provide new information for comparing the gene maps of ruminants, humans and mice. The deer hybrids are an important new resource that can contribute to the comparative analysis of the mammalian genome.
Assuntos
Mapeamento Cromossômico , Cervos/genética , Hibridização Genética , Animais , Evolução Biológica , Sondas de DNA/genética , Feminino , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de Restrição , Especificidade da EspécieRESUMO
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)
Assuntos
Fatores de Crescimento Neural/metabolismo , Ovário/fisiologia , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Maturidade Sexual , Animais , Autorradiografia , Feminino , Expressão Gênica , Imuno-Histoquímica , Radioisótopos do Iodo , Peso Molecular , Ovário/crescimento & desenvolvimento , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Receptores de Fator de Crescimento Neural , Células Tecais/citologia , Células Tecais/fisiologiaRESUMO
Growth factors are commonly associated with the regulation of cellular proliferation and differentiation. In established cells, growth factors can also serve as trophic agents. Immortalized LHRH neurons contain basic fibroblast growth factor (bFGF) receptors. Although these receptors are coupled to activation of protein kinase C, and phorbol esters are strong activators of protein kinase C-stimulated LHRH release, bFGF did not influence LHRH secretion from these cells. To clarify this discrepancy, the effects of bFGF and phorbol ester on pro-LHRH biosynthesis, protein processing, and secretion were examined in GT1-7 cells. Phorbol ester stimulated LHRH secretion, whereas bFGF either had no effect or stimulated LHRH release depending upon the antiserum used. Pro-LHRH levels in lysate and medium were depressed by phorbol esters; concentrations in bFGF-treated cells were somewhat lower than those in unstimulated controls. HPLC analyses revealed that both agents enhanced the release of LHRH intermediate products into the medium. C-Terminally extended forms of LHRH, especially LHRH-[Gly11], were prominent in medium from bFGF-stimulated neurons. Levels of LHRH were depressed relative to those in the control or phorbol ester groups. These data indicate that phorbol esters control the biosynthesis, secretion, and, to some extent, processing of pro-LHRH. The effects of bFGF are novel because this factor regulates processing of the prohormone so that LHRH-intermediate products are predominantly secreted instead of LHRH. By enhancing the secretion of these intermediates over that of LHRH, bFGF can control the biological activity of the decapeptide and regulate LHRH neuronal function.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Precursores de Proteínas/biossíntese , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The rat ovary is innervated by sympathetic nerve fibers. Since the development and survival of peripheral sympathetic neurons innervating nonreproductive organs have been shown to depend on the production of nerve growth factor (NGF) by the innervated tissues, the present experiments were undertaken to determine if the immature rat ovary has the capability of synthesizing NGF. Blot hybridization of ovarian polyadenylated RNA (A+-RNA) to a NGF cRNA probe revealed the presence of a 1.3- to 1.4-kilobase (kb) mRNA species similar to mature NGF mRNA detected in mouse submaxillary gland, a source rich in NGF. Quantitation of NGF protein by a sensitive and specific two-site enzyme immunoassay demonstrated the presence of NGF in juvenile ovaries at levels comparable to those found in other sympathetically innervated tissues. Neither denervation of the ovary nor treatment with gonadotropins (hCG and FSH) or somatomammotropins (PRL and GH) affected the levels of NGF mRNA. However, denervation significantly increased NGF levels, suggesting that, as in other target tissues, denervation prevents the retrograde transport of NGF by the sympathetic terminals and leads to accumulation of the protein at its site of production. It is concluded that 1) the developing ovary is able to both transcribe the NGF gene and translate its mRNA into NGF protein; and 2) the NGF content in the ovary is regulated by its innervation. The results provide the biochemical basis for the concept, elaborated in the companion paper, that NGF through its trophic actions on ovarian sympathetic neurons contributes to the regulation of ovarian development and, hence, to the acquisition of female reproductive capacity.
Assuntos
Regulação da Expressão Gênica , Hormônios/farmacologia , Fatores de Crescimento Neural/genética , Ovário/fisiologia , Animais , Autorradiografia , Denervação , Feminino , Hipofisectomia , Ovário/crescimento & desenvolvimento , Ovário/inervação , RNA Mensageiro/metabolismo , Ratos , Sistema Nervoso Simpático/metabolismoRESUMO
TrkA, the nerve growth factor (NGF) tyrosine kinase receptor, is expressed not only in the nervous system, but also in nonneural cells, including discrete cellular subsets of the endocrine and immune system. In the rat ovary, trkA receptor abundance increases strikingly in thecal-interstitial cells during the hours preceding the first ovulation. Blockade of either trkA transducing capacity or NGF biological activity inhibited ovulation, suggesting a role for NGF in the ovulatory process of this species. To identify some of the processes that may be affected by trkA activation in the thecal compartment, we used purified thecal cells/thecal fibroblasts from bovine ovaries (heretofore referred to as thecal cells). Ribonuclease protection assays employing bovine-specific cRNA probes demonstrated the presence of the messenger RNAs (mRNAs) encoding NGF and its receptors, p75 NTR and trkA, in the thecal compartment of small, medium, and large antral follicles and showed that trkA mRNA is also expressed in granulosa cells. In situ hybridization and immunohistochemical examination of intact ovaries confirmed these cellular sites of NGF and trkA synthesis. TrkA mRNA, but not NGF mRNA, was lost within 48 h of placing thecal cells in culture. Thus, to study trkA-mediated actions of NGF on these cells we transiently expressed the receptor by transfection with a vector containing a full-length rat trkA complementary DNA under transcriptional control of the cytomegalovirus promoter. Because ovulation is preceded by an LH-dependent increase in androgen and progesterone production, the ability of NGF to modify the release of these steroids was determined in freshly plated cells still containing endogenous trkA receptors and in cells undergoing luteinization in culture that were transiently transfected with the trkA-encoding plasmid. NGF stimulated both androgen and progesterone release in freshly plated thecal cells, but not in luteinizing cells provided with trkA receptors. As ovulation in rodents requires an increased formation of PGE2 and has been shown to be antedated by proliferation of thecal fibroblasts, we determined the ability of NGF to affect these parameters in trkA-transfected thecal cells. The neurotrophin rapidly stimulated PGE2 release and amplified the early steroidal response to hCG in trkA-expressing cells, but not in cells lacking the receptor. Likewise, NGF stimulated [3H]thymidine incorporation into trkA-containing cells, but not into cells that had lost the receptor in culture. Induction of ovulation in immature rats by gonadotropin treatment verified that an increased cell proliferation in the thecal compartment, determined by the incorporation of bromodeoxyuridine into cell nuclei, occurs 4-5 h before ovulation in this species. These results suggest that the contribution of NGF to the ovulatory process includes a stimulatory effect of the neurotrophin on steroidogenesis, PGE2 formation, and proliferative activity of thecal compartment cells.
Assuntos
Fator de Crescimento Neural/farmacologia , Folículo Ovariano/citologia , Células Tecais/efeitos dos fármacos , Androstenodiona/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Ciclo-Oxigenase 2 , Dinoprostona/metabolismo , Feminino , Expressão Gênica , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/genética , Fator de Crescimento Neural/genética , Ovulação/efeitos dos fármacos , Progesterona/metabolismo , Prostaglandina-Endoperóxido Sintases/genética , Sondas RNA , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptor de Fator de Crescimento Neural/genética , Receptor trkA/genética , Receptor trkA/fisiologia , Receptores de Fator de Crescimento Neural/genética , Ovinos , Células Tecais/citologia , Células Tecais/fisiologiaRESUMO
Activation of trkA, the nerve growth factor (NGF) tyrosine kinase receptor, has been recently implicated in the process of mammalian ovulation. During the hour preceding follicular rupture, a marked increase in trkA and NGF gene expression occurs in thecal-interstitial cells of the ovary. Immunoneutralization of NGF actions or pharmacological blockade of trkA transducing activity inhibits ovulation, suggesting that activation of the NGF-trkA complex in nonneural cells of the periovulatory follicle is a physiological component of the ovulatory cascade. As thecal cells of Graafian follicles are functionally coupled by gap junctions, and the ovulatory rupture requires dissociation of thecal cell-cell communication, we sought to determine whether NGF affects the integrity of this communication. We now report that NGF-induced activation of trkA receptors in isolated ovarian thecal cells disrupts cell to cell communication by affecting the functional integrity of gap junctions. Bovine thecal cells expressing trkA receptors, but not cells lacking the receptors, respond to NGF with a reduction in the transfer of calcein, a fluorescent dye that passes through gap junctions. This effect was associated with a rapid (10-30 min) increase in serine phosphorylation of connexin-43, the main protein constituent of gap junctions in the ovary. The reduction in dye transfer was not observed when the cells were exposed to epidermal growth factor or other neurotrophins, including neurotrophin 3, neurotrophin 4, and brain-derived neurotrophic factor. Thus, cell-specific activation of trkA receptors in periovulatory follicles may provide one of the signals involved in inducing the cellular dissociation of the follicular wall that precedes ovulatory rupture.
Assuntos
Comunicação Celular , Junções Comunicantes/fisiologia , Fatores de Crescimento Neural/fisiologia , Ovulação/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Células Tecais/fisiologia , Animais , Bovinos , Comunicação Celular/efeitos dos fármacos , Conexina 43/química , Conexina 43/metabolismo , Feminino , Fatores de Crescimento Neural/farmacologia , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Ovário/metabolismo , Fosforilação , Receptor trkA/metabolismoRESUMO
A single injection of estradiol valerate induces a form of cystic ovary resembling some aspects of the human polycystic ovarian syndrome. Preceding the development of follicular cysts, there is an increase in intraovarian synthesis of nerve growth factor (NGF) and the low affinity NGF receptor (p75 NGFR). Selective blockade of NGF actions and p75 NGFR synthesis in the ovary restored estrous cyclicity and ovulatory capacity in estradiol valerate-treated rats, suggesting that an increase in NGF-dependent, p75 NGFR-mediated actions within the ovary contributes to the development of cystic ovarian disease. We have tested this hypothesis by grafting NGF-producing neural progenitor cells into the ovary of juvenile rats that have been induced to ovulate precociously by a single injection of PMSG. The NGF-producing cells, detected by their content of immunoreactive p75 NGFR material, were found scattered throughout the ovary with some of them infiltrating the granulosa cell compartment of large, precystic follicles. Ovarian NGF content was 2-fold higher than in the ovary of rats receiving control cells. Estrous cyclicity was disrupted, with the animals showing prolonged periods of persistent estrus, and an almost continuous background of vaginal cornified cells at other phases of the estrous cycle. Morphometric analysis revealed that the presence of NGF-producing cells neither reduced the total number of corpora lutea per ovary nor significantly increased the formation of follicular cysts. However, the ovaries receiving these cells showed an increased incidence of precystic, type III follicles, accompanied by a reduced number of healthy antral follicles, and an increased size of both healthy and atretic follicles. These changes in follicular dynamics were accompanied by a selective increase in serum androstenedione levels. The results show that an abnormally elevated production of NGF within the ovary suffices to initiate several of the structural and functional alterations associated with the development of follicular cysts in the rat ovary.
Assuntos
Androgênios/metabolismo , Estro/efeitos dos fármacos , Fatores de Crescimento Neural/fisiologia , Ovário/fisiologia , Androgênios/sangue , Androstenodiona/metabolismo , Animais , Transplante de Células/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Técnicas de Transferência de Genes , Imuno-Histoquímica , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Síndrome do Ovário Policístico/sangue , Síndrome do Ovário Policístico/metabolismo , Radioimunoensaio , Ratos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Testosterona/metabolismo , Fatores de TempoRESUMO
Several members of the neurotrophin (NT) family, including nerve growth factor (NGF), NT-3, and NT-4/5, are expressed in the mammalian ovary. As their respective receptor tyrosine kinases are also found in the gland, the possibility exists that NTs act directly on the gonads to exert effects unrelated to their support of the ovarian innervation. We now report that trkA, the NGF receptor tyrosine kinase, is involved in the acute activational process that leads to the first ovulation. The trkA gene becomes transiently expressed in periovulatory follicules at the time of the first preovulatory surge of gonadotropins at puberty; the increase in trkA messenger RNA (mRNA) content is dramatic ( > 100-fold), but transient (approximately 9 h). No such changes in trkB or trkC mRNA were observed; the abundance of these mRNAs, which encode the receptor tyrosine kinase for NT-4/5 and brain-derived neurotrophic factor, and NT-3, respectively, remained at very low levels throughout puberty. In vivo and in vitro experiments demonstrated that the activation of trkA gene expression is brought about by the proestrous discharge of LH. The increase in trkA mRNA levels is mainly localised to cells of the follicular wall and interstitial tissue of the ovary. NGF mRNA abundance also increases at proestrus, with peak values detected about 5 h before ovulation; as in the case of trkA mRNA, NGF mRNA was found in thecal-interstitial cells. Both trkA and NGF protein, detected by immunohistochemistry, were localized to this same ovarian compartment. Interleukin-1 beta (IL-1 beta), a putative mediator of LH action, enhances both trkA and NGF gene expression in ovarian cells, an effect prevented by IL-1ra, a natural IL-1 beta receptor antagonist. Il-1 beta also stimulates PGE2 release, and this effect was inhibited by both NGF antibodies and a trk receptor blocker, NGF antibodies administered in vivo attenuated the increase in ovarian PGE2 synthesis that antedates ovulation. Immunoneutralization of NGF action or pharmacological blockade of trk tyrosine kinase activity targeted to one ovary resulted in the ipsilateral inhibition of ovulation. The remarkably narrow time frame of trkA gene activation at the completion of follicular growth suggests that NGF acting as a neuroendocrinotrophic factor in a developmentally restricted manner contributes to the acute cytodifferentiation process that leads to the first ovulation in mammals.
Assuntos
Ovulação/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo , Células Cultivadas , Feminino , Gonadotropinas Equinas/farmacologia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/genética , Neurotensina/genética , Ovário/citologia , Ovário/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/genética , Receptor do Fator Neutrófico Ciliar , Receptor trkA , Receptor trkC , Receptores de Fator de Crescimento Neural/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Prosencéfalo/fisiologia , Maturidade Sexual/genética , Fatores de Transcrição/genética , Transcrição Gênica , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Hipotálamo Anterior/fisiologia , Hipotálamo Anterior/efeitos da radiação , Cinética , Mamíferos , Dados de Sequência Molecular , Fator 2 de Transcrição de Octâmero , Área Pré-Óptica/fisiologia , Área Pré-Óptica/efeitos da radiação , Regiões Promotoras Genéticas , Prosencéfalo/crescimento & desenvolvimento , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
It is becoming increasingly evident that the secretory activity of LHRH neurons is regulated not only by transsynaptic inputs but also by trophic molecules of glial and neuronal origin. The present experiments were undertaken to gain insights into the potential cell-cell mechanisms by which basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF alpha), two growth factors produced in the hypothalamus, may affect LHRH neuronal function. Northern blot analysis showed that the LHRH-producing cell line GT1-7 contains the messenger RNA (mRNA) encoding the type 1 fibroblast growth factor receptor (FGFR-1) but not that encoding the epidermal growth factor (EGF) receptors, which mediates the biological actions of both TGF alpha and EGF. Ligand-induced receptor phosphorylation experiments demonstrated that GT1-7 cells possess biologically active FGFR-1s but not EGF receptors. Exposure of the cells to bFGF resulted not only in FGFR-1 tyrosine phosphorylation, but also in tyrosine phosphorylation of phospholipase C gamma, one of the initial enzymes in the intracellular signaling cascade initiated by FGFR activation. GT1-7 cells proliferated in response to this activation. Despite the presence of biologically active receptors, bFGF did not significantly stimulate release of the mature LHRH decapeptide. Instead, bFGF increased the steady-state levels of the mRNA encoding the LHRH precursor processing endoprotease PC2, with a time course comparable to that of phorbol esters, suggesting that, as shown in the companion paper, the actions of the growth factor on LHRH neurons involve facilitation of the initial step in LHRH prohormone processing. The increase in PC2 gene expression was not accompanied by changes in LHRH mRNA levels. Unlike these direct actions of bFGF on GT-1 cells, TGF alpha appears to act indirectly via astroglial intermediacy. Exposure of GT1-7 cells to TGF alpha or EGF failed to affect several parameters of cellular activity including LHRH release, LHRH and PC2 mRNA levels, and cell proliferation. In contrast, astrocyte culture medium conditioned by treatment with TGF alpha led to sustained stimulation of LHRH release with no changes in LHRH gene expression and a transient increase in PC2 mRNA levels. Although no definitive evidence for the presence of FGFR-1 in normal LHRH neurons could be obtained by either double immunohistochemistry or double in situ hybridization procedures, fetal LHRH neurons in primary culture responded to bFGF with neurite outgrowth. Thus, normal LHRH neurons may have an FGFR-1 content too low for detection by regular histochemical procedures, and/or detectable expression of the receptor may be confined to a much earlier developmental stage. The mitogenic effect of bFGF on GT1-7 cells supports this possibility and suggests a role for FGF in the cell proliferation events that precede acquisition of the LHRH neuronal phenotype. It appears that once this phenotype is established, bFGF may promote the differentiation of LHRH neurons. The results also suggest that the secretory capacity of LHRH neurons develops under a dual trophic influence, one on peptide processing exerted directly by bFGF on early neurons, and another on LHRH release, exerted by TGF alpha via the intermediacy of astroglial cells.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Substâncias de Crescimento/farmacologia , Neuroglia/fisiologia , Neurônios/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/fisiologia , Animais , Divisão Celular , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/análise , Receptores ErbB/genética , Receptores ErbB/fisiologia , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Pró-Proteína Convertase 2 , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Subtilisinas/genética , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
We have isolated ovine follistatin cDNA from an ovarian follicle cDNA library and determined its sequence. The deduced amino acid sequence of the ovine follistatin precursor is highly homologous (greater than 97%) to the porcine, human and rat follistatins. Northern analysis was used to characterize follistatin gene expression in ovaries of adult ewes, collected from days 11 to 13 of the oestrous cycle. Two major (about 2.7 kb and 1.5 kb) and one minor (about 0.5 kb) transcripts were detected in polyadenylated RNA extracted from ovarian follicles and corpora lutea. The degree of expression of the transcripts varied in the two ovarian compartments, with the 2.7 kb species predominating in the follicles and the 1.5 kb species being more abundant in the corpora lutea. No transcripts were detected in stromal tissue containing preantral follicles of less than 1 mm.
Assuntos
Glicoproteínas/genética , Fase Luteal/fisiologia , Ovário/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA/isolamento & purificação , Feminino , Folistatina , Glicoproteínas/biossíntese , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , OvinosRESUMO
The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.
Assuntos
Colífagos/genética , Proteínas de Ligação a DNA/genética , Sequência de Aminoácidos , Sequência de Bases , Colífagos/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , Proteínas de Ligação a DNA/metabolismo , Genes Virais , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
Nerve growth factor (NGF), its messenger RNA (mRNA) and its receptor protein and mRNA are found in several brain regions. Little attention has been given to the possibility that the hypothalamus, which controls the endocrine system, may produce NGF and/or express NGF receptors. This would indicate the existence in this brain area of neuronal populations which are either target for NGF-responsive cells or sensitive to NGF, respectively. Blot hybridization of polyadenylated (poly(A)+) RNA to a mouse NGF cRNA probe revealed the presence of NGF mRNA in both the suprachiasmatic region (henceforth, called preoptic area [POA]) and medial basal hypothalamus (MBH) of developing female rats. The mRNA was already detectable on fetal day 18 and reached maximal levels around postnatal day 3 (MBH) and 12 (POA), declining thereafter. This pattern was temporally different than that of areas known to produce NGF, i.e., the neocortex (Cc) and hippocampus (Hc). In agreement with previous reports, NGF mRNA levels in these areas were negligible before birth, became maximal between the second and third week of postnatal life and decreased moderately thereafter. The concentration of NGF protein, measured by a two-site enzyme immunoassay, was 3 times higher in the POA than in the MBH at an infantile age (day 12), increasing 2-fold in the POA of juvenile animals (day 28) but not in the MBH. This developmental pattern was similar to that seen in the Hc, though the NGF concentrations were significantly lower in the POA.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Hipotálamo/crescimento & desenvolvimento , Fatores de Crescimento Neural/biossíntese , Receptores de Superfície Celular/biossíntese , Animais , Química Encefálica , Feminino , Regulação da Expressão Gênica , Genes , Hipotálamo/embriologia , Hipotálamo/metabolismo , Fatores de Crescimento Neural/genética , Hibridização de Ácido Nucleico , Poli A/análise , RNA/genética , RNA Complementar , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos , Receptores de Superfície Celular/genética , Receptores de Fator de Crescimento NeuralRESUMO
The report of endorphin-like activity in human CSF [7] has stimulated us to study peptides in various CSF specimens. At first we attempted to fractionate CSF by gel-filtration and paper-mapping procedures. By these techniques, we have isolated a peptide (Gly-Ala3-Val-Leu) contaminated with glutamic acid (or glutamine), which could not be removed by either of these methods. An HPLC method was developed for peptide fractionations on a RP-18 column with a gradient of 18 mM ammonium acetate and acetonitrile. This system has been evaluated with synthetic peptides of molecular weight up to about 1850, and has the advantages that the buffer salts may be removed by lyophilization and separations are performed at or near neutral pH thus minimizing alterations in the structure of the peptides. The retention characteristics of peptides on a C18 column is a function of the pH of the buffer. This facilitates the characterization of peptides since each may be identified by its unique chromatographic behavior at different pH values on one column.
Assuntos
Peptídeos/líquido cefalorraquidiano , Aminoácidos/análise , Cromatografia em Gel/métodos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Papel/métodos , HumanosRESUMO
Cell-free preparations from superior and inferior colliculi of very young rats (1-3 days old) contained adenylate cyclase systems which were highly responsive to serotonin. The response to serotonin declined markedly during early development and was very low at maturity. Adenylate cyclase activity in the 10,000 times g particulate fraction from colliculi of newborn rats was significantly stimulated by 0.05 muM serotonin. Half-maximal activation was produced with less than 1 muM serotonin. Maximal stimulation of collicular adenylate cyclase was about 80% above basal enzyme activity and occurred with approximately 50 muM serotonin. Tryptamine and several derivatives of serotonin produced responses which were comparable to that obtained with serotonin; 5-methoxytryptamine was uniformly the most active compound tested. Norepinephrine or dopamine produced much smaller increases in adenylate cyclase activity. Stimulation of collicular adenylate cyclase by serotonin was significantly but incompletely blocked by serotonin antagonists, including d-lysergic acid diethylamide (d-LSD), 2-bromo-d-lysergic acid diethylamide, methysergide, 1-methyl-8 beta-carbobenzyloxy-aminomethyl-10 alpha-ergoline and cyproheptadine. Chlorpromazine also produced partial blockade. In contrast, l-lysergic acid diethylamide, haloperidol, propranolol, phenoxybenzamine and morphine were ineffective as serotonin blocking agents. Of the compounds which produced a partial blockage of serotonin action, d-LSD, cyproheptadine and chlorpromazine were themselves capable of stimulating adenylate cyclase activity. These results are consisent with the existence of multiple receptors in rat brain which are capable of interacting with indoleamines.