Figure 1.

GT1-7 cells express NK3R and respond to agonist with altered GnRH secretion and transcription. A, RT-PCR for NK3R using total RNA from GT1-7 cells and mouse brain. A single band of the expected 387 bp size was observed in cDNA prepared with reverse transcriptase (RT) but not negative controls prepared without reverse transcriptase (No RT). Bands were excised and confirmed to be NK3R by sequencing. B, GnRH peptide accumulation in conditioned media from GT1-7 cells. Cells were treated with 50 nM senktide or vehicle for either three or 24 hours. GnRH peptide content in conditioned media was measured by RIA. Data are normalized to vehicle control and shown as mean fold ± SEM for n = 3 to 5 independent experiments. Comparisons were made using a t-test against the hypothetical mean 1.0. *Significantly different from 1.0 (P < .05). C, Transient transfection of the –5 Kb GnRH gene sequence upstream of a luciferase reporter (-5 Kb GnRH-Luc) into GT1-7 cells treated with vehicle or senktide (1 or 10 nM) for 10 hours. Luciferase values were internally normalized using a cotransfected β-galactosidase reporter to control for transfection efficiency and normalized to an external control. Data are represented as mean ± SEM for n = 3 independent experiments. Statistical analysis was performed using ANOVA with Least squares means differences Tukey HSD (α = .05). Levels not connected by same letter are significantly different.