In vitro exposure of porcine granulosa cells to the phytoestrogens genistein and daidzein: Effects on the biosynthesis of reproductive steroid hormones

Abstract

Since a discrepancy concerning the effects of phytoestrogens on steroidogenesis exists in the literature we investigated the effects of genistein and daidzein on progesterone and estradiol synthesis in cultured primary granulosa cells derived from follicles of porcine ovaries. In this context, the investigation was performed to test the hypothesis that isoflavones can reduce hydroxysteroid dehydrogenase/isomerase (3β-HSD) activity by down-regulation of its transcription. We found that daidzein did not impair the viability of cultured granulosa cells in the concentration range from 0.1 to 100 μM, but genistein inhibited the cell viability at 50 μM compared to the unexposed controls. Forskolin (10 μM) and pregnenolone (2.5 μM) enhanced the basal progesterone secretion in the absence of both phytoestrogens. Daidzein or genistein at non-toxic concentrations alone or combined with forskolin or pregnenolone significantly reduced progesterone synthesis. This reduction was not due to changes of the abundance of P450scc protein, but the gene hydroxysteroid dehydrogenase/isomerase ( 3β-HSD) was significantly decreased at a non-toxic concentration of daidzein (50 μM) in non-stimulated and pregnenolone-stimulated cells. Moreover, genistein (1, 10 μM) significantly inhibited the 3β-HSD-mRNA only in pregnenolone-stimulated granulosa cells. It can be suggested that the effect of genistein on steroidogenesis only partly results from the impairment of 3β-HSD gene expression. In non-toxic concentrations daidzein and genistein did not change the androstenedione- or testosterone-stimulated estradiol-17β synthesis. In summary, genistein and daidzein have direct effects on porcine granulosa cell progesterone synthesis which involve the inhibition of 3β-HSD enzyme activity across the post-cyclic AMP pathway.