Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

Abstract These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys 6 ] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys 6 ] GnRH exhibited a higher affinity and was 7–10 × more potent than unlabeled [D-Lys 6 ] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys 6 ] GnRH. The GnRH stain was followed by immunocytochemical stains for LHβ, FSHβ or 25–39 ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60–90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90–100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.