Figure 4.

GnRH Induction of FSHβ mRNA and FSHβLUC Activity Is Attenuated at Both High and Low GnRH Pulse Frequencies by PKA Inhibition. LβT2 cells were transiently transfected with (A) 2 μg −140/+15 FSHβLUC or (B) 2 μg −797/+5 LHβLUC, as well as 2 μg of empty vector (EV, black solid bars) or DNPKA (DN, white solid bars). After 20 hours of perifusion with high (ΛΛΛΛ; every 30 min) or low (Λ__Λ; every 120 min) frequencies of GnRH or medium alone (____), luciferase activity was measured. Endogenous (C) FSHβ mRNA levels or (D) LHβ mRNA levels were quantified by real-time RT-PCR in LβT2 cells transfected with 2 μg of empty vector (EV, black solid bars) or 2 ug of DNPKA (DN, white solid bars) followed by 20 hours of perifusion with high (ΛΛΛΛ; every 30 min) or low (Λ__Λ; every 120 min) frequencies of GnRH or medium alone (____). Bar graphs show relative change compared with basal control samples (mean ± SEM from three independent experiments each performed in triplicate, normalized to β-galactosidase activity (A and B) or endogenous ribosomal protein L19 (RPL19) mRNA levels (C and D)). Significant differences (P < .05), measured by one-way ANOVA with a post hoc Tukey multiple-comparison test, are indicated by different letters.