Endocytosis of follicle-stimulating hormone by ovarian granulosa cells: analysis of hormone processing and receptor dynamics

Suspensions of freshly isolated rat granulosa cells were used to study endocytosis and processing of radioiodinated ovine follicle-stimulating hormone (I-oFSH) and to analyze the dynamics of its receptor. Ovine FSH was iodinated to a specific activity of 26 microCi/micrograms as determined by radioreceptor self-displacement assays with maximum specific binding to excess membrane receptors of 46%. Radiolabeled oFSH was judged biologically equivalent to the unlabeled hormone since I-oFSH shows saturation-binding kinetics and stimulates steroidogenesis in a similar dose-related manner to unlabeled oFSH. Experiments designed to study the extent and time course of degradation involved continuous exposure of isolated granulosa cells to I-oFSH. Saturation of membrane receptors was achieved within 1.5 h of incubation, and internalization of FSH occurred in a linear manner for up to 6 h. The rate of internalization was equivalent to 2,780 FSH molecules/cell/h. Degradation of FSH became apparent after 6 h of incubation and increased to 86% of total cellular-associated radioactivity at 22 h. FSH degradation was inhibited by 100 microM chloroquine or 0.45 mM leupeptin. The measurement of cell surface I-oFSH binding in the combined presence of 100 microM chloroquine and 0.5 mM cycloheximide was unchanged for up to 22 h of incubation. This and other receptor binding data suggest that there is no reutilization of FSH receptors. Scatchard analyses of 4 degrees C binding assays on intact cells indicated that a two-site model best fit the data with association constants of K11 = 1.44 (+/- .42) X 10(10) and K12 = 4.35 (+/- .91) X 10(8). Receptor binding and activation studies for progesterone production yielded ED50s of 270 pM and 7.7 pM, respectively, and also indicated that 20% receptor occupancy is sufficient to stimulate maximal progesterone production. We conclude that after the initial binding event, FSH is endocytosed very slowly and is subsequently shuttled to the lysosomal compartment for degradation. The retarded rate of endocytosis may relate to novel pathways of hormone processing.