LAUSR

Abstract Many local factors expressed in the ovary are involved in folliculogenesis. Among these, the ovarian renin-angiotensin system is critical for normal processes of follicle development, steroidogenesis and ovulation. In various clinical conditions such as secondary hypertension, heart failure, and liver and/or renal damages, hyperaldosteronism can be detected. In addition, Aldo levels are known to be high in patients with PCOS and/or OHSS. However, the effect of high concentration of Aldo on reproductive function has not been fully elucidated. In this study, we examined the influence of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells, by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. Granulosa cells isolated from female rats were treated with follicle stimulating hormone (FSH) in the presence of low to high concentrations of Aldo (1-300 nM) and cultured in serum-free conditions. After incubation with Aldo for 48 hours, changes in estradiol (E2) and progesterone (P4) productivity induced by FSH stimulation were measured by CLIA, and cAMP synthesis was evaluated by EIA. Total RNA was extracted and mRNA level of steroidogenetic enzymes induced by FSH and that of BMP-receptors and Id-1 induced by BMP-6 were quantified by real-time RT-PCR. Phosphorylation of Smad1/5/9 was further evaluated by Western blotting using cell lysate after the co-treatments with Aldo and BMP-6. As a result, treatment with Aldo increased FSH-induced P4 production by granulosa cells in a concentration-dependent manner at the maximum effects by 100-300 nM of Aldo. Aldo treatment increased mRNA levels of P4-synthetic enzymes expression including StAR, P450scc and 3βHSD, and Aldo also upregulated FSH-induced cAMP synthesis. On the contrary, Aldo failed to change E2 production stimulated by FSH. Moreover, Aldo treatment attenuated Id-1 transcription and phosphorylation of Smad1/5/9 induced by BMP-6 stimulation. These data indicated that Aldo elicits a specific effect on the enhancement of P4 production by granulosa cells. On the other hand, BMP-6 treatment downregulated MR expression in granulosa cell, suggesting the existence of a negative feedback loop between BMP-receptor and MR signaling. Taken it into consider that BMP ligands act as specific suppressor for P4 production as a luteinizing inhibitor, it is possible that Aldo is functionally linked to the suppression of endogenous BMP-receptor signaling in granulosa cells, leading to the deterioration of follicular growth and ovulation process. Collectively, it is revealed that high concentration of Aldo specifically enhances FSH-induced P4 production by inhibiting BMP signal in granulosa cells. This effect may be associated with impaired fertility and/or ovulatory failure in the patients with hyperaldosteronism.