Transforming Growth Factor-β 1 Inhibits Ovarian Androgen Production: Gene Expression, Cellular Localization, Mechanisms(s), and Site(s) of Action*

Abstract

It is the aim of this study to establish ovarian transforming growth factor-.beta.1 (TGF.beta.1) gene expression, to reevaluate its cellular localization, and to explore potential interactions of this regulatory peptide on ovarian androgen biosynthesis. Northern analysis of whole ovarian polyadenylated RNA revealed a single 2.5-kilobase transcript corresponding to the TGF.beta.1 precursor. Immunohistochemical staining localized the protein to the thecal-interstitial (interfollicular) compartment. To explore potential autocrine effects of TGF.beta.1, use was made of whole ovarian dispersates from immature rats the differentiation of which was monitored by the acquisition of androgen biosynthetic capacity. The accumulation of androsterone, the major androgenic steroid detectable in this culture system, increased 5.4-fold over baseline in response to treatment with hCG (1 ng/ml). This effect was further optimized (2- to 4-fold) by supplementation with insulin (1 .mu.g/ml) and insulin-like growth factor-I (50 ng/ml). In the absence of these optimizing supplements. TGF.beta.1 (10 ng/ml) was without effect on basal androsterone accumulation, producing distinct, albeit relatively limited (25%), inhibition of hCG hormonal action. In contrast, supplement-mediated optimization of ovarian androgen biosynthesis revealed TGF.beta.1 to be a highly potent inhibitor (>80%) of hCG hormonal action. This reversible TGF.beta.1 action proved time and dose dependent, with a minimal time requirement of 72 h and a median inhibitory dose of 2.6 ng/ml. TGF.beta.1 action was not due to diminution in the viable cell mass or altered cAMP generation and, therefore, most likely involved a site(s) of action distal to or independent of cAMP generation. Cellular radiolabeling studies of TGF.beta.-treated ovarian cells disclosed the accumulation of steroid intermediates proximal to the 17.alpha.-hydroxylation step, suggesting TGF.beta.1-mediated blockade at the level of the steroidogenic enzume 17 .alpha.-hydroxylase/17-20-lyase. Taken together, these observations are in keeping with the view that TGF.beta.1, possibly of thecal-interstitial origin, may not only play a positive paracrine role at the level of the adjacent granulosa cell (as previously reported), but may also constitute one of several autocrine signals concerned with the regulation of ovarian androgen economy. As such, these findings reaffirm the polyfunctional nature of TGF.beta.1 action, as manifested by its diametrically opposed effects in different ovarian compartments.