CHARACTERIZATION OF GONADOTROPIN-RELEASING-HORMONE GENE TRANSCRIPTS IN A MOUSE HYPOTHALAMIC NEURONAL GT1 CELL-LINE

We have characterized the nuclear and cytoplasmic RNA transcripts deri ved from the gonadotropin releasing hormone (GnRH) gene in a mouse hyp othalamic neuronal GT1 cell line. Analyses of nuclear GnRH RNA precurs ors present in the GT1 cells by RNase protection assay show that there is no particular order of intron excision, suggesting the existence o f multiple processing pathways. A similar pattern is observed in mouse preoptic area-anterior hypothalamus (POA-AH). In GT1 cells, approxima tely 5% of the total GnRH RNA transcripts are found in the nucleus. In contrast, in the POA-AH of mice, nuclear transcripts comprise 40% of the total GnRH transcripts. Thus the GT1 cells, while similar in overa ll GnRH RNA processing to mouse hypothalamic GnRH neurons, do not exhi bit the high abundance of nuclear GnRH RNA transcripts seen in the rod ent GnRH neuron in vivo. Quantitative analysis of the nuclear RNA spec ies shows that the GnRH primary transcript comprises more than 90% of the total nuclear GnRH mRNA precursors in both GT1 cells and mouse POA -AH and thus GnRH processing intermediates account for fewer than 10% of these precursors. Using these probes, we have examined changes in G nRH primary transcript expression in GT1-7 cells. In the presence of R NA synthesis inhibitors, the half-life of the GnRH primary transcript was found to be quite short, similar to 18 min, suggesting that the le vel of primary transcript would reflect levels of GnRH gene transcript ion. When GT1-7 cells are treated with the phorbol ester PMA (phorbol, 12-myristate, 13-acetate) for 1 h, GnRH primary transcript levels dec rease by similar to 70%. Supporting the hypothesis that GnRH primary t ranscript is a good indicator of GnRH gene transcription is the findin g that 1 h of PMA treatment results in a similar (similar to 50%) decr ease in GnRH gene transcription, as assayed by nuclear run-on assay. O ur observation that GT1 cells resemble mouse hypothalamic GnRH neurons in their pattern of intron excision and in the ratio of primary trans cript to other nuclear transcripts emphasizes the utility of these cel ls for studying the regulation of GnRH gene expression in this immorta lized hypothalamic cell line.