Regulation of Gonadotropin Receptors and Steroidogenesis in Cultured Porcine Leydig Cells*

Abstract

Cultures of porcine Leydig cells (>80% pure) were used to study the regulation of LH/hCG receptors and Leydig cell responsiveness over several days. These cells contained approximately 60,000 receptors/cell, with an apparent K_d of 1.5 × 10^-9 M. Binding of hCG to receptor was time and dose dependent (saturation at 4–6 h), and maximal binding was maintained for 24 h. After removal of hCG, radioactivity was released into the medium. This spontaneous release (50% released by 8 h) was unaffected by the presence of cold hormone in the medium. About 80% of the released material was inactive in a binding assay and had the properties of monoiodotyrosine. Throughout the period of culture, hCG receptor was maintained at or above the original level. The addition of hCG during the culture caused a dose-and time-dependent down-regulation of receptor. After removal of the hormone, hCG receptors increased, reaching 50% of control within 24 h. Most of the bound radioactive hormone could be released by a brief acid treatment (pH 4 for 4 min). At 15 min of binding, 80% of the hormone could be released, with a decrease to less than 30% released by 2 h of binding. The acid-releasable [¹²⁵I]iodo-hCG remained constant from 2–8 h of binding even though the total cell-associated radioactivity increased until 6–8 h. After saturation binding, the cells were allowed to release the bound radioactivity. These cells were rechallenged with radioactive hormone and were capable of rebinding hormone, although not to the level originally attained. These data syggested that in a culture system, the hCG receptors in Leydig cells reached a steady state turnover rate where internalization of gonadotropin receptors was balanced by the appearance of new receptors on the cell surface. Cycloheximide caused an increase in hCG binding, suggesting that receptor synthesis was not the limiting or controlling step in the steady state turnover at this early time period.