Radioligand-Receptor Assay of Luteinizing Hormone and Chorionic Gonadotropin

Abstract

The specific binding of labeled gonadotropin to subcellular fractions of the rat testis has been utilized in the development of a sensitive radioligand assay system for LH and HCG. The cell-free supernatant of the fragmented interstitial cell fraction was found to contain high affinity gonadotropin receptors, which showed rapid and specifically reversible binding of ¹²⁵I-labeled LH and HCG. For binding assays, satisfactory results were also obtained with the fraction prepared by centrifugation at 1500 g after homogenization of the whole testis. Assays were performed by incubation of tracer hormone with standards or samples and binding fraction for 2 hr at 37 C, or 16 hr at 24 C. Separation of bound and free tracer was performed by centrifugation and washing of the particulate receptor fraction, followed by measurement of the bound radioactivity. Parallel dose-response curves were obtained with human pituitary LH (LER 907), human urinary LH, and human chorionic gonadotropin, while ovine, bovine, and rat LH gave parallel standard curves of slightly lower slope than that of the human hormones. Satisfactory standard curves for LH and HCG were obtained over the range 10-500 mU. The presence of LH-HCG receptors with similar characteristics has also been demonstrated in the rat ovary. Radioligand-receptor gonadotropin assays based on binding to gonadal cell fractions are extremely rapid and sensitive, combining the convenience and precision of binding assays with the biological specificity characteristic of the receptor site.