Specificity of the immunocytochemical luteinizing hormone-releasing hormone receptor reaction

Affinity-purified anti-LHRH, affinity-purified LHRH-anti-LHRH complex, as well as antisera 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 as well as purified antibody conferred moderate immunocytochemical staining to the secretion granules of rat pituitary gonadotrophs. Staining intensity became greatly increased upon treatment of the electron microscopic 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. It is concluded that gonadotroph secretion granules contain, in addition to an LHRH receptor reacting with LHRH in vitro, stable complexes of endogenous LHRH with receptor.