Localization of biotinylated gonadotropin releasing hormone on pituitary monolayer cells with avidin-biotin-peroxidase complexes

Abstract

The new avidin-biotin-peroxidase complex (ABC) technique was used to localize the [D-Lys6] analog of gonadotropin releasing hormone (GnRH), labeled with biotin, on pituitary monolayer cultures from female rats. Staining was diffuse, or in patches, on the surface of 10-17% of the cells 30 sec-3 min after the addition of 10(-10)-10(-12) M biotin-labeled GnRH. In parallel studies, double stains for gonadotropins showed label on 16.3 +/- 2% of the monolayers. Capping was evident by 3 min after exposure and the stain appeared in dense patches, vesicles, or granules 10-30 min after exposure. The stain was abolished by the addition of a 10- to 100-fold excess of unlabeled [D-Lys6] GnRH. Biotinylated GnRH released luteinizing hormone (LH) and follicle stimulating hormone (FSH) and was either equipotent or 10 times more potent than the unlabeled analog in multiple dose-response tests. The ED50 of the 4 hr release was 0.075 nM for LH and 0.02 nM for FSH. Competitive binding assays showed that the binding affinity of the biotinylated GnRH was within the range found for the unlabeled analog (0.7 nM-IC50). This report describes the localization of biotinylated GnRH on the surfaces of cells exposed to low concentrations of the analog with a technique that requires minimal manipulation of the cells, and is performed in less than one day.