TRANSFORMING GROWTH-FACTOR-BETA INHIBITS OVARIAN 17-ALPHA-HYDROXYLASEACTIVITY BY A DIRECT NONCOMPETITIVE MECHANISM

Transforming growth factor-beta 1 (TGF-beta 1) inhibits theca-intersti tial cell (TIC) androgen biosynthesis while enhancing progesterone pro duction without altering P450(17 alpha) protein content. The purpose o f the present study was to define the mechanism of TGF-beta 1 inhibiti on of ovarian androgen production by determining the effects of TGF-be ta 1 on steroidogenic enzyme messenger RNA (mRNA) expression and 17 al pha-hydroxylase activity in TIC in vitro. TIC isolated from hypoph yse ctomized immature rat ovaries by Percoll gradient centrifugation were cultured with and without LH and TGF-beta 1 up to 6 days. At various t imes, cytoplasmic mRNA was extracted from the TIC, and P450(SCC), SP-H SD and P450(17 alpha) mRNA were measured by specific assays, using RT- PCR. Treatment with TGF-beta 1 alone (0.1-100 ng/ml) had no effect on mRNA expression at 2 days but increased P450(SCC) and 3 beta-HSD mRNA at 4 days. TGF-beta 1 did not alter the LH stimulation of P450(SCC) an d 3 beta-HSD mRNA up to 6 days but caused a modest (2.5-fold) increase in P450(17 alpha) mRNA at 2 days. Specificity studies with inhibin-A( 30 ng/ml), activin-A (100 ng/ml), and MIS (300 ng/ml) demonstrated tha t the effects of TGF-beta 1 were unique within this family of peptides . We next examined the effect of TGF-beta 1 on 17 alpha-hydroxylase ac tivity. Kinetic analysis revealed that the 17 alpha-hydroxylase enzyme has an apparent Michaelis-Menten constant of 3.42 mu mol/liter and ma ximum velocity of 0.23 pmol/min mg protein. TGF-beta 1 inhibited 17 al pha-hydroxylase activity by a noncompetitive mechanism with an apparen t inhibin constant (K-i) of 46.4 pM. The results of our studies demons trate that TGF-beta 1 directly inhibits TIC androgen production by a n oncompetitive mechanism. This novel mechanism may be important in prev enting excessive androgen production in developing ovarian follicles w ithout preventing differentiation of the TIC.