Dissociation of hCG-receptor complex and detection of hCG receptors in human chorionic tissues

We developed a radioreceptor assay (RRA) system for investigating occupied hCG receptors by using a rat testis membrane fraction. Treatment with dithiothreitol, neuraminidase, and low pH (3.3) buffer partially dissociated hCG from its receptor with dissociation rates of 42.8%, 35.2%, and 68.7%, respectively. Specific receptor rebinding was unaffected and showed competitive inhibition of [125I]hCG rebinding by unlabeled hCG. Scatchard plot analysis yielded a rebinding Km of 1.798-3.496/pM. High concentrations of NaCl and MgCl2 had no obvious effect on the [125I]hCG-receptor complex. Triton X-100 and high concentration MnCl2 treatments resulted in the loss of the receptor rebinding activity. We also investigated human chorionic tissue (at 8 weeks, 11 weeks, 16 weeks, 24 weeks, and term) for hCG receptors using the above method. Treatments with different agents increased [125I]hCG binding in this tissue preparation 139-226%, but Scatchard analysis failed to demonstrate any specificity. [125I]hCG uptake by the choriocarcinoma BeWo cell-line increased 151-193% after similar treatments, but this increase could not be competitively inhibited by unlabeled hCG. Treatment with dithiothreitol, neuraminidase, and low pH (3.3) buffer seems feasible for detecting hCG-occupied receptors. Our results suggest that there are no hCG receptors distributed on the surface of the syncytiotrophoblast of human placenta.