Exogenous Secreted Frizzled-Related Protein-4 Modulates Steroidogenesis of Rat Granulosa Cells through Wnt/β-catenin and PI3K/AKT Signaling Pathways

BACKGROUND: It has been reported that secreted frizzled-related protein-4 known as an antagonist of Wnt signaling pathway plays a role in luteinization process of rodent granulosa cells. The purpose of this study was twofold: 1) to determine whether recombinant human secreted frizzled-related protein-4 (rhSFRP-4) could directly induce terminal differentiation of rat Granulosa Cells (GCs) and 2) to understand how the modulation of β-catenin and Protein Kinase B (PKB)/AKT activity by exogenous SFRP-4 could be involved in steroidogenesis. METHODS: GCs were firstly stimulated with Follicle-Stimulating Hormone (FSH) named as FSH-primed cells then were treated with luteinizing hormone (LH). Then estradiol (E(2)) and progesterone (P(4)) production levels were assessed in the absence or presence of rhSFRP-4 treatment. The expression levels of activated β-catenin, pAKTser ( 473 ) , pGSK3βser ( 9 ) were assessed by western blot or immunofluoresence. RESULTS: In the presence of rhSFRP-4, there was 38% decreased E(2) levels compared to untreated FSH-primed cells (p<0.05), and P(4) production subsequently decreased. However, in GCs pre-treated with rhSFRP-4 prior to addition of FSH, P(4) levels increased 2-fold compared with untreated cells (p<0.05). Unexpectedly, treatment with rhSFRP-4 prior to LH stimulation inhibited LH-induced P(4) secretion. Treatment with low (0.5 ng/ml) but not high (50 ng/ml) concentrations of rhSFRP-4 led to significantly increased levels of pGSK3βser ( 9 ) (1.6-fold) and nuclear active β-catenin (2.8-fold) in GCs compared with untreated cells. Interestingly, pre-treating GCs with rhsFPR4 prior to LH stimulation resulted in a 38% decrease in pAKTser ( 473 ) levels compared with those in LH-treated cells (p<0.05). CONCLUSION: Taken together, our results showed that rhSFRP-4 could directly induce terminal differentiation in GCs via the modulation of β-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner.