RESUMEN
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.
Asunto(s)
Expresión Génica , Hormona Liberadora de Gonadotropina/genética , Smegmamorpha/metabolismo , Animales , Química Encefálica , Cromatografía Líquida de Alta Presión , Ciprinodontiformes/metabolismo , Femenino , Hormona Liberadora de Gonadotropina/aislamiento & purificación , Masculino , Hipófisis/química , Extractos de Tejidos/químicaRESUMEN
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.
Asunto(s)
Encéfalo/metabolismo , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/farmacología , Secuencia de Aminoácidos/genética , Animales , Cricetinae , Femenino , Hormona Folículo Estimulante/metabolismo , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/genética , Hormona Liberadora de Gonadotropina/aislamiento & purificación , Hidroxiprolina/análogos & derivados , Hidroxiprolina/farmacología , Hipotálamo/metabolismo , Hormona Luteinizante/metabolismo , Masculino , Espectrometría de Masas , Mesocricetus , Hipófisis/citología , Hipófisis/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.
Asunto(s)
Encéfalo/metabolismo , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/aislamiento & purificación , Hipófisis/metabolismo , Precursores de Proteínas/metabolismo , Animales , Argentina , Química Encefálica , Cromatografía Líquida de Alta Presión , Peces , Hormona Liberadora de Gonadotropina/análisis , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/metabolismo , Hipófisis/química , Precursores de Proteínas/análisis , Precursores de Proteínas/química , Radioinmunoensayo , Extractos de Tejidos/químicaRESUMEN
The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (cIGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.