RESUMO
Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.
Assuntos
Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Smegmamorpha/metabolismo , Animais , Química Encefálica , Cromatografia Líquida de Alta Pressão , Ciprinodontiformes/metabolismo , Feminino , Hormônio Liberador de Gonadotropina/isolamento & purificação , Masculino , Hipófise/química , Extratos de Tecidos/químicaRESUMO
Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.
Assuntos
Encéfalo/metabolismo , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Sequência de Aminoácidos/genética , Animais , Cricetinae , Feminino , Hormônio Foliculoestimulante/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/isolamento & purificação , Hidroxiprolina/análogos & derivados , Hidroxiprolina/farmacologia , Hipotálamo/metabolismo , Hormônio Luteinizante/metabolismo , Masculino , Espectrometria de Massas , Mesocricetus , Hipófise/citologia , Hipófise/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
The neuropeptide GnRH is the major regulator of reproduction in vertebrates acting as a first signal from the hypothalamus to pituitary gonadotropes. Three GnRH molecular variants were detected in the brain of a fish, pejerrey (Odontesthes bonariensis), using chromatographic and immunological methods. The present study shows that one form is identical to chicken GnRH-II (sequence analysis and mass spectrometry) and the second one is immunologically and chromatographically similar to salmon GnRH. The third form was proven to be a novel form of GnRH by isolating the peptide from the brain and determining its primary structure by chemical sequencing and mass spectrometry. The sequence of the novel pejerrey GnRH is pGlu-His-Trp-Ser-Phe-Gly-Leu-Ser-Pro-Gly-NH(2), which is different from the known forms of the vertebrate and protochordate GnRH family. The new form of GnRH is biologically active in releasing gonadotropin and GH from pituitary cells in an in vitro assay.
Assuntos
Química Encefálica , Peixes/metabolismo , Hormônio Liberador de Gonadotropina/química , Aminoácidos/análise , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Hormônio Liberador de Gonadotropina/síntese química , Hormônio Liberador de Gonadotropina/farmacologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/isolamento & purificação , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Radioimunoensaio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The presence and distribution of gonadotropin-releasing hormone (GnRH) has been investigated in the Mexican leaf frog, Pachymedusa dacnicolor, brain during development and in the adult. The ontogenetic pattern of GnRH neurons illustrates their extracranial as well as intracranial sites. Immunohistochemical analysis indicates that GnRH-immunoreactive neurons appear during the metamorphic climax. They are located in the mesencephalon and subsequently other GnRH neurons appear in the peripheral terminal nerve and anterior preoptic area of the brain. Use of specific antisera and homologous combined with heterologous preabsorption tests indicate that mammalian and chicken GnRH-II-like peptide-containing neurons are differentially located within the brain, the former in the anterior preoptic area and peripheral terminal nerve and the latter in the mesencephalon. HPLC and RIA data suggest the presence of three forms of immunoreactive GnRH in the P. dacnicolor brain. A mammalian GnRH-like molecule and a chicken GnRH-II-like molecule are present. A third form, suspected to be [hydroxyproline9]mGnRH elutes before the mammalian GnRH.