The orphan nuclear receptor, steroidogenic factor 1 (SF-1), plays a major role in adrenal and gonadal development, as well as in sexual differentiation. It has been demonstrated that the expression of a number of genes regulated by SF-1 is inhibited by the transforming growth factor, (TGF-beta). To date, however, the influence of TGF-beta on the expression of SF-1 gene has not been reported. A Northern blot analysis with the use of a radiolabeled cDNA probe, and immunodetection with antibodies directed against SF-1, demonstrated that the Sf-1 transcript and the SF-1 protein levels were lowered by TGF-beta in Y-1 adrenocortical cells, both in untreated and adenylyl cyclase activator, forskolin-treated cells. An examination of the Sf-1 transcript stability in the presence of actinomycin D revealed no influence of TGF-beta on the rate of Sf-1 mRNA decay. Inhibition of Sf-1 expression by TGF-beta was abolished by cycloheximide, suggesting that the growth factor inhibitory effect requires ongoing protein synthesis. We conclude that in Y-1 cells TGF-beta inhibits the expression of SF-1 gene at a transcriptional level, and we postulate that the inhibitory effect of TGF-beta on steroid hormone synthesis in the adrenal cortex could be due to an attenuated transcription of Sf-1.

Polycystic ovarian syndrome (PCOS) is a prevalent endocrine disorder, which is the most common cause of anovulatory infertility and hirsutism. It is caused by an overproduction of androgens in theca cells. In the ovary, androgen synthesis is regulated by 17 alpha-hydroxylase/17,20-lyase enzyme complex containing P450c17 (CYP17). In some individuals, the promoter region of CYP17 gene contains a T-->C substitution that creates an Sp1 site at position-34. This polymorphism generates a recognition site for the MspA1 restriction enzyme. The objective of the study was to investigate the frequency of T-->C substitution of CYP17 gene promoter in women with PCOS and elucidate its role in the pathogenesis of the syndrome. Another aim of the study was to compare the results with the levels of the hormones luteinizing hormone, follicle-stimulating hormone, testosterone and estradiol. The present investigation involved a group of 55 women with PCOS and 56 healthy women without symptoms of PCOS. Following digestion with restriction enzyme MspA1, it was demonstrated that the polymorphic A2 allele is no more frequent in women with PCOS than in healthy women. In the PCOS group, the hormonal profiles were not significantly different in the carriers of a normal allele A1A1 from the carriers of A1A2 and A2A2 alleles. It is concluded that T-->C polymorphism of CYP17 gene is not associated with steroid hormone synthesis in PCOS and is not the primary genetic defect in this disease.

The expression of negative regulators of CYP17 gene expression: DAX-1, COUP-TF and N-CoR was investigated in bovine adrenocortical cells in primary culture. The cells were incubated for 6 hours in a defined medium, containing ACTH (10 nM) or forskolin (25 microM). Total RNA was isolated, the concentration of CYP17 gene transcript was determined by hybridization analysis, and polyA RNA was reverse-transcribed to obtain cDNA. Fragments of CYP17, DAX-1, COUP-TF, N-CoR and beta-actin cDNA were amplified by PCR and the amplified cDNAs were analyzed by agarose gel electrophoresis followed by densitometry. It was found that DAX-1 and COUP-TF mRNA levels were unaffected by treatment with ACTH or forskolin. On the contrary, both compounds enhanced the level of mRNA encoding N-CoR. Under the same conditions, ACTH or forskolin substantially stimulated CYP 17 mRNA accumulation. Our results suggest that in the adrenocortical cells in culture, the expression of DAX1 and COUP-TF is constitutive, whereas N-CoR expression might be inducible.

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