Fate of a Gonadotropin-Releasing Hormone Agonist Internalized by Rat Pituitary Gonadotrophs*

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Abstract
The intracellular fate of a radiolabeled GnRH agonist ([D-Ala6]des-Gly10-GnRH ethylamide) taken up by rat pituitary gonadotrophs in vivo was determined by quantitative electron microscopic autoradiography. Rats ovariectomized for 2 weeks were given intracarotid injections of the ligand, and pituitary glands were removed, counted, and processed for electron microscopic autoradiography at specific intervals thereafter. Uptake of the GnRH agonist by the pituitary was specific, since simultaneous administration of the radiolabeled peptide and a 200-fold excess of the unlabeled peptide inhibited binding by greater than 90%. A simple grain density (GD) analysis of autoradiograms revealed six cell compartments to be significantly labeled (GD > 1): plasma membrane, vesicles, lysosomes, Golgi elements, nuclear membrane, and secretory granules. Four of these (plasma membrane, vesicles, lysosomes, and Golgi elements) showed distinct changes in the pattern of labeling with time: 5 min after the injection, the greatest concentration of label (9.1) was seen over the plasma membranes of gonadotrophs, but vesicles (5.5) and lysosomes (2.3) were already significantly labeled. At subsequent intervals plasmalemmal labeling decreased and that of vesicles, lysosomes, and Golgi elements increased. Labeling of vesicles peaked at 60 min (9.9), whereas the concentration of label over Golgi (1.8) and lysosomes (36.3) was highest at 120 min. The high concentration of grains over lysosomes indicates accumulation of label in this compartment. The nuclear membrane and secretory granules were significantly labeled (∼1.5 and ∼4.0, respectively) at all time points, with no change in labeling over time. The cytoplasm (0.5), mitochondria (0.6), and endoplasmic reticulum (0.3) were not significantly labeled at any time point. Results of thin layer electrophoresis of label extracted from pituitaries at different times revealed that at 5, 15, and 30 min, there was one major species with a mobility greater than that of the intact hormone but equivalent to that of label extracted from serum. At 60 min three products were demonstrated, one of which was identified as monoiodotyrosine. These findings are compatible with the conclusion that the GnRH agonist binds to the surface of the gonadotrophs, is taken up by receptor-mediated endocytosis, and is transported both to the Golgi complex and to lysosomes, where it accumulates and is degraded.

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