Specificity of the Immunocytochemical Luteinizing Hormone-Releasing Hormone Receptor Reaction

Abstract

Affinity-purified anti-LHRH [luteinizing hormone-releasing hormone], affinity-purified LHRH-anti-LHRH complex, and antisera [rabbit] depleted of anti-LHRH by solid phase immunoabsorption were used to test the specificity of the immunocytochemical approach for detection of receptor-bound LHRH. The solid phase immunoabsorbent was prepared by attaching LHRH to Sepharose via an RNase spacer. Purified anti-LHRH was eluted from the immunoabsorbent by acidification. Unabsorbed antisera and purified antibody conferred moderate immunocytochemical staining to the secretion granules of rat pituitary gonadotrophs. Staining intensity became greatly increased on treatment of EM sections with LHRH before immunocytochemical staining. All gonadotroph staining was abolished when absorbed antisera were used. The specificity of the immunoabsorbent was tested on mixtures of anti-LHRH and anti-ACTH17-39. On absorption of such mixtures with insolubilized LHRH, all gonadotroph staining disappeared, but the optical density indices of ACTH staining remained unaffected. Gonadotroph secretion granules contain, in addition to an LHRH receptor reacting with LHRH in vitro, stable complexes of endogenous LHRH with receptor.