‘In Situ’ Characterization of Gnrh Receptors: Use of Two Radioimagers and Comparison with Quantitative Autoradiography

Abstract

New radioimagers, the HRRI (high resolution radioimager) and the Phosphorimager (phosphor screen: PS), apt to display more ample linear dose-response scale than radio-sensitive films, were tested in comparaison with quantitative autoradiography (QA). GnRH receptor saturation experiments were achieved on tissue sections (rat pituitary, rat brain, human ovary) with a iodinate GnRH agonist (¹²⁵l-[D-Ala⁶, Des-Gly¹⁰]-LH-RH Ethylamide) for determination of affinity constant (K_d). In rat pituitary, comparable results were obtained with the 3 methods (K_d: 0.4 to 0.6 nM). Discrepancies occured in the hippocampus and in the granulosa cell layer of the preovulatory follicle, due to low resolutive (PS) or short linear dose-response (films) performances. In the hippocampus GnRH receptor affinity was under-estimated with PS (K_d: 2.3 vs 0.5 and 0.6 nM for QA and HRRI respectively). In the follicular granulosa cell layer it was over-estimated by QA (0.5 vs 50 nM for the HRRI), while PS did not allow resolution of this thin cell layer. In conclusion, the HRRI is a very powerful tool for the quantification of in situ radioligand binding (binding sites study and in situ hybridization) in very discrete areas.