RESUMEN
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 beta-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 beta-catenin, pAKTser[473], pGSK3 beta ser[9] were assessed by western blot or immuno-fluoresence
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 pGSK3 beta ser[9] [1.6-fold] and nuclear active beta-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 beta-catenin and PKB/AKT pathways and that it does so in a dose-dependent manner