RESUMEN
Many studies on Gnrh1, and the teleost Gnrh3, have elucidated the roles of these peptides in reproductive regulation. However, the role of the midbrain population of Gnrh, Gnrh2, has long been a mystery, despite its ubiquitous conservation in all jawed vertebrates except rodents. Previous behavioral studies in sparrows, musk shrews, mice, zebrafish, and goldfish show that Gnrh2 administrations both increase spawning behaviors and decrease feeding behaviors, suggesting a role of this peptide in metabolism regulation along with the canonical role in regulating reproduction. In order to more deeply explore the roles of Gnrh2, we used a cyprinid teleost, zebrafish, which has 2 forms of Gnrh, Gnrh2 and Gnrh3, to generate a knockout zebrafish line which contains a frameshift mutation and subsequent disruption of the coding for the functional Gnrh2 peptide. We examined differences in reproduction, feeding, growth, and mobility in this line, and discovered major differences in feeding and growth parameters, suggesting that Gnrh2 is a potent anorexigen in zebrafish. Additionally, there were no differences in mobility except for increased distances swam during feeding periods. There were no major differences in reproductive success, however, female gnrh2-/- zebrafish exhibited smaller oocytes and increased embryo mortality, indicating slightly decreased oocyte quality. Additionally, there were changes in the expression levels of many feeding, growth, and reproductive neuropeptides in gnrh2-/- zebrafish. Taken together, these findings suggest a role for Gnrh2 in controlling satiation in zebrafish along with a minor role in maintaining optimal oocyte quality in females.
Asunto(s)
Conducta Alimentaria , Técnicas de Inactivación de Genes , Hormona Liberadora de Gonadotropina/metabolismo , Oocitos/metabolismo , Pez Cebra/metabolismo , Animales , Peso Corporal , Femenino , Hormona Liberadora de Gonadotropina/deficiencia , Gónadas/metabolismo , Reproducción/fisiología , Factores de TiempoRESUMEN
We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.