The Binding of Iodinated hCG to Porcine Granulosa Cells1

Abstract

Intact, viable porcine granulosa cell suspensions have been employed in an in vitro system to make direct observations of specific gonadotropin interactions with ovarian tissue. Radioiodinated hCG (125I—hCG) was incubated with these cell suspensions. Specific binding to cells of the 125I—hCG was directly proportional to both cell number and 125I—hCG concentration, although at higher concentrations of 125I—hCG it was possible to saturate the cells with labeled hormone. Approximately 1,000 molecules of 125I—hCG were bound/cell after 10 min of incubation at 37 C. The binding was greater at 37 than at 0 C and increased with time at both temperatures. Specificity of this 125I–hCG binding was indicated by the observation that 125I–BSA bound to granulosa cells less than 125I–hCG, and that monkey kidney and HeLa cell suspensions bound less 125I–hCG than granulosa cells. Increasing concentrations of unlabeled hCG in the incubation mixture progressively inhibited the uptake of 125I–hCG by granulosa cells. LH, either of human, porcine or ovine origin, was also able to significantly compete for 125I–hCG binding. FSH had little effect and probably binds at different receptor sites in this system. The binding of desialyzed hCG in this system was quite similar to that of intact hCG, and this supports the conclusion that sialic acid is not required on hCG for receptor recognition. A 100–fold molar excess of the individual pure α and β subunit preparations of hCG had no competitive effect on 125I—hCG binding, which suggests that neither subunit alone is sufficient to convey significant receptor affinity. Non—gonadotropic hormones, ACTH and bovine growth hormone, had little effect. (Endocrinology91: 65, 1972)