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Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-beta superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 microM) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3 beta HSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3 beta HSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1.5-fold) and activity (twofold) but with minimal effects on the mRNA for 3 beta HSD and P450scc. HOTT cell responses to activin (0.05-50 ng/ml) and inhibin (0.05-50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome.

Aromatase cytochrome P-450 (P-450AROM) is the enzyme in the steroidogenic pathway controlling the formation of oestrogens from 19 carbon steroid precursors. Aromatase is present in various tissues of the human fetus. The liver is second only to the placenta in the level of P-450AROM activity in the fetus. In this study we examined the effects of type 1 transforming growth factor-beta (TGF beta) on P-450AROM expression in human fetal (HF) hepatocytes. The HF hepatocytes were dispersed into single cells which were placed into monolayer cell culture until confluent. Cells were then rinsed and treated in serum-free media with dibutyryl cyclic AMP (Bu2cAMP) for 72 h. Treatment with Bu2cAMP (2 mmol/l) caused a fivefold increase in aromatase activity in hepatocytes. The increase in aromatase activity apparently represented an increase in P-450AROM enzyme as determined by immunoblotting using an antibody directed against human placental aromatase. TGF beta blocked basal, as well as Bu2cAMP increases, in aromatase activity by over 50%. The effect of TGF beta was dose-dependent with maximal inhibition observed using 2-5 ng TGF beta/ml. Immunodetectable P-450AROM decreased in parallel with activity following TGF beta treatment. The mechanism of TGF beta action was not through increasing phosphodiesterase (PDE) breakdown of cAMP since inhibition of PDE had no effect on TGF beta action. Finally we examined the level of P-450AROM mRNA using competitive polymerase chain reaction amplification. Bu2cAMP increased mRNA levels of P-450AROM by 2.5-fold, while TGF beta inhibited this induction by 35%. The results of this investigation demonstrated that TGF beta is a potent regulator of P-450AROM expression in HF hepatocytes.

The interconversion of oestrone and oestradiol, androstenedione and testosterone, and dehydroepiandrosterone and 5-androstene-3 beta,17 beta-diol in mammalian tissues is catalysed by 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR). To identify tissue sites of 17 beta-HSOR activity in the human fetus, microsomal fractions from 15 different fetal tissues obtained from first and second trimester pregnancies were used for evaluation of enzymatic activity by use of [17 alpha-3H] oestradiol as the substrate and NADP+ as the co-factor. With these reagents, the enzyme-catalysed reaction led to the production of both non-radiolabelled oestrone and NADP3H in equimolar amounts; the radioactivity associated with NADP3H was used to quantify 17 beta-HSOR activity. Activity of 17 beta-HSOR was present in microsomes of all the tissues evaluated. The specific activity of the enzyme was highest in liver and placental microsomes. The interconversion of oestradiol and oestrone in microsomal fractions of nine different fetal tissues was studied by the use of substrates labelled with tritium at stable nuclear positions ([6,7-3H]oestradiol and [6,7-3H]oestrone). The products, [3H]oestrone and [3H]oestradiol, were quantified by the use of established techniques; other metabolites formed in these incubations were not identified. The reductive pathway of metabolism (oestrone to oestradiol) appeared to be favoured in microsomal fractions prepared from placenta, fetal zone of the adrenal gland and, possibly, lung. The oxidative pathway (oestradiol to oestrone) appeared to be favoured in microsomes prepared from liver, intestine, stomach, kidney, brain and heart. 17 beta-HSOR activity in fetal liver also was assessed by the use of fresh and frozen-thawed tissue, homogenate, subcellular fractions, and, also, in primary hepatocytes maintained in culture; the specific activity of the enzyme was highest in the microsomal fraction of liver tissue and 17 beta-HSOR activity in liver microsomes was linear with time of incubation up to 1 h. In hepatocytes, the enzymatic activity was linear with time of incubation up to 2 h and with cell number up to 2.5 x 10(5) cells/ml; the apparent Michaelis constant of hepatocyte 17 beta-HSOR for oestradiol was 11 mumol/l. The specific activity of 17 beta-HSOR did not change after pretreatment of hepatocytes for 24 h with insulin, glucagon or dexamethasone.