Pulsatile GnRH stimulation increases steady-state mRNA levels for FSHbeta, LHbeta, and alpha subunits in superfused...

The pituitary secretion of FSH and LH is controlled by pulsatile GnRH stimulation of specific magnitude and frequency, and changes in the mode of stimulation can dramatically alter the pattern of gonadotropin secretion. We explored the effects of different GnRH treatments on the secretion of gonadotropins and levels of mRNA for the gonadotropin subunits in superfused cultures of immature rat pituitary cells. After 4 days of stationary culture in the absence of GnRH, the cells were exposed during superfusion to different GnRH treatments for up to 20 h. Each GnRH pulse (6 min/h, 10 nM) elicited pulsatile release of both gonadotropins, causing a 2- to 3-fold increase of mean hourly secretion of FSH and 5- to 6-fold increase of LH. The magnitude of gonadotropin responses after 4, 10, and 20 h of stimulation with GnRH pulses was not significantly different. In contrast, mRNA levels for different subunits varied depending on the duration of GnRH exposure. Only FSHbeta mRNA was increased (2.5 +/-0.4-fold,P < 0.05) after 4 h of stimulation, and only after 10 h was the a subunit mRNA also significantly increased (2.1 +/- 0.3-fold, P < 0.05). After 20 h of GnRH exposure, both mRNA levels became further elevated (4-to 7-fold vs no GnRH) and the LHbeta mRNA increased significantly (5.7 +/- 0.9-fold, P < 0.05). The effects of continuous (20 h) exposure to GnRH on mRNA levels were different from those of pulsatile treatment. A stimulatory effect comparable to that of pulsatile stimulation was observed only for the a subunit, whereas FSHbeta mRNA was significantly reduced (P < 0.05 vs no GnRH) and LHbeta mRNA level was not affected. These studies demonstrated, for the first time, that mRNA steady-state levels for all three gonadotropin subunits can be up-regulated by GnRH in vitro, but following different time intervals, and that the mode and duration of GnRH treatment can profoundly affect the results. Our results also indicate that caution must be exercised when interpreting data from experiments conducted in vitro with continuous or short-term GnRH treatments.