Fig. 7. Nab2 and Dax1 Inhibit GnRH Pulse Sensitivity of the LHβ Promoter.

LβT2 cells cultured on microcarrier beads as in Fig. 1 were transfected with the −1.8 kb LHβ or −398 bp FSHβ promoter reporter vectors and serum starved for 16 h. Cells were then perifused while pulsed with vehicle or 100 nm GnRH at 120- or 30-min pulse intervals. After 6 h, cells were harvested and assayed for reporter vector activity. A, The LHβ promoter exhibits a pulse-dependent increase in activity that is not exhibited by the FSHβ promoter. #, Significant difference (P ≤ 0.05) between 120- and 30-min pulse. B, Cotransfection with Nab2 or Dax1 blocks frequency-dependent activation of the LHβ promoter. Cells transfected as above except with the addition of pcDNA3 null expression vector or vector encoding Nab2 or Dax1 were serum starved 16 h and perifused for 6 h while pulsed with vehicle or 10 nm GnRH at 120-min or 30-min pulse intervals. Luciferase activity normalized to non-pulsed null vector control is plotted. Mean values are plotted with error bars representing sem. *, Significant difference (P ≤ 0.05) of Dax1-transfected, vehicle-pulsed cells from pcDNA3 transfected vehicle-pulsed cells. #, Significant difference (P ≤ 0.05) between Nab2- and Dax1-transfected, 30-min pulsed cells from 30-min null vector-transfected cells. Both Dax1 and Nab2 attenuated the activation at 30-min pulse intervals, and Nab2 had no effect on activation at 120-min pulse intervals. V, Vehicle.