Thyroid hormone inhibits aromatase activity in porcine thecal cells cultured alone and in coculture with granulosa cells.

We cultured porcine thecal and granulosa cells alone or in coculture to define whether thyroid hormone affects aromatase activity in porcine ovarian cells. Dispersed cells were cultured with 10(-9) M triiodothyronine (T3) for 24 hours. Testosterone (final concentration 10(-7) M) was added as aromatase substrate for granulosa cells (Gc) cultured alone. Thecal (Th) androgens serve as a substrate for estradiol secretion by Th cells cultured alone and in coculture with Gc. At the end of the preincubation time, the culture media was removed and replaced with fresh media containing 100 ng follicle stimulating hormone (FSH) or 10(-3) M 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP). After overnight incubation, the culture media was analyzed for estradiol production by radioimmunoassay (RIA). T3 inhibited basal, FSH-stimulated, and 8brcAMP-stimulated estradiol production in all culture conditions. T3 inhibited cAMP analogue 8-Br-cAMP and FSH-induced aromatase activity to a similar extent, thus suggesting that the inhibitory effect of T3 is downstream of cAMP formation. In the second part of the experiment a rabbit polyclonal antibody against human placental cytochrome P-450arom was used to confirm the effect of T3 on aromatase protein in Th and Gc. Pretreatment of Th and Gc with T3 markedly decreased immunostaining for aromatase in both types of cells, suggesting a direct effect of T3 on this enzyme.