IN-SITU HYBRIDIZATION DETECTION OF TSH-BETA SUBUNIT GENE-EXPRESSION IN THE SERUM-FREE PRIMARY CULTURE OF THE ADULT-RAT PITUITARY

This study was designed to construct the primary culture system to det ect the change in TSH beta subunit (TSH beta) gene expression in indiv idual cells. Adult, male Wistar rats were sacrificed by transcardial p erfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3 or 6 days with or without 1 nM triiodothyron ine (T-3) under the serum-free condition. In some cultures, TRH was ad ded to a final concentration of 1 mu M on 6, 12 or 24 h before fixatio n. Then the culture media were removed to measure TSH concentration. C ells were fixed with paraformaldehyde and hybridized with S-35-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography wa s subsequently performed. T-3 treatment markedly suppressed relative c ellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatme nt on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH con centration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, a nd 24 h on day 6. On day 2 and 3, although T-3 treatment suppressed ba sal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T-3 treatment. These results show that the thyroid h ormone and TRH regulate TSH beta gene expression independently. Our cu lture system may provide a useful model to examine the action of indiv idual substances on a specific subpopulation of the anterior pituitary cells.