Protein synthesis is required for testosterone to decrease ornithine decarboxylase messenger RNA levels in rat Sertoli cells.

We have previously shown that pretreatment of Sertoli cells, derived from 21-day-old Wistar rats, with 5 x 10(-7) M testosterone decreases ornithine decarboxylase (ODC) mRNA levels. In the present study we examined the mechanism of this inhibition. Pretreatment of Sertoli cells for 48 h with 5 x 10(-7) M testosterone decreased ODC mRNA levels 30% at all time points examined. Addition of 25 micrograms/ml cycloheximide (CHX) to the testosterone-pretreated cells resulted in a loss of the testosterone-mediated decrease in ODC mRNA levels by 4 h. Surprisingly, a further 1.8-fold increase in ODC mRNA was observed at 8 h compared to that in untreated cells. Addition of CHX to control cells also resulted in a measurable increase in ODC mRNA levels by 4 h, with a further 1.6-fold increase at 8 h. Identical effects were observed using the protein synthesis inhibitor puromycin (200 micrograms/ml), which has a mechanism of action different from that of CHX. Analysis of the half-life of ODC mRNA after actinomycin-D (5 micrograms/ml) inhibition demonstrated that testosterone did not alter ODC mRNA half-life compared to that in control cells (2.63 vs. 2.55 h). These results demonstrate that the mechanism by which testosterone decreases ODC mRNA levels requires continual protein synthesis, since the effect can be abolished by treating the cells with CHX or puromycin. The data also demonstrate that the testosterone effect is exerted primarily at the transcriptional level, since the half-life of ODC mRNA was not affected by testosterone.