Morphological Characterization of Immortalized Hypothalamic Neurons Synthesizing Luteinizing Hormone-Releasing Hormone

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An immortalized LHRH cell line has recently been developed by genetically targeting these neurons for tumorigenesis. One of the subclones, the GT1-7 cells, was characterized at both the light and electron microscopic levels to study the cellular and subcellular organization of these cells, particularly as they relate to biosynthesis, processing, and secretion. The cells were fixed onto slides 18-36 h after plating. LHRH and GnRH-associated peptide (GAP) immunoreactivities (IR) were detected by immunocytochemistry using colloidal gold labeling. These cultured cells exhibited the classical neuronal appearance of LHRH neurons, and they established numerous interconnections. Neighboring neurons were coupled by tight junctions, while more distant cells were interconnected with neural axon-like processes and collaterals. This cellular organization is suggestive of a neural network where neuronal activity is coordinated. At the ultrastructural level, the nondividing cells possessed indented nuclei, well developed Golgi complexes, and abundant numbers of ribosomes and secretory granules. Clathrin-coated vesicles were found in fusion with the plasma membrane. The ribosomes and secretory vesicles were particularly prominent, suggestive of high rates of protein biosynthesis and secretion. All of the cells immunostained for both LHRH and GAP; however, GAP IR was always more pronounced than that for LHRH. This finding was corroborated by biochemical data reported in a companion paper. The GAP IR was associated with ribosomes and secretory vesicles. By comparison, LHRH IR was restricted mainly to the secretory vesicles. Using colloidal gold particles of different sizes to denote LHRH or GAP IR, it was determined that both GAP and LHRH IR were colocalized within the same secretory vesicle. Taken together, these data suggest that pro-LHRH is biosynthesized on the ribosomes, packaged as an intact protein into the secretory vesicles, processed to LHRH and GAP-(1-56) within these vesicles, and transported to the periphery of the cell in preparation for secretion. These morphological data emphasize the utility of using these immortalized LHRH neuronal cells to dissect the cellular and subcellular architecture involved in biosynthesis, processing, and secretion. In addition, our results provide the first detailed evidence for the intracellular pathway involved in pro-LHRH biosynthesis, processing, and secretion in these cultured neuronal cells.