P-58: Secreted Frizzeled Related Protein Type-4as an Inducer of Apoptosis and Terminal Differentiationof Rat Granulosa Cells

Background: Involvement of Wnt proteins and one of its antagonist known as secreted Frizzled Related Protein type-4 (sFPRP-4) was reported in rodent ovarian follicular development. Other studies showed an ap- Abstracts of the 11th Royan International Congress on Reproductive Biomedicine 7 7 International Journal of Fertility & Sterility (IJFS), Vol 4, Suppl 1, Summer 2010 optotic-associated expression of sFRP-4 and also additionalnon apoptotic-related function such as relationship with luteinization events in rat corpus luteum. This study sought to determine whether sFRP4 could be a direct inducer of apoptosis or terminal differentiation in rat granulosa cells by using recombinant human sFRP4 (rhsFRP4). To this order, the effect of rhsFRP-4 on subcellular localization of beta-catenin as an important mediator in Wnt/beta-catenin signaling and steroidogenesis was further examined. Materials and Methods: Immature female rats were stimulated with PMSG (10 IU), ovaries were removed after 48h and granulosa cells were isolated mechanically. Cells were cultured in the presence of Testosterone (0.1 nM) and recombinant human FSH (50 ng/ml) for 48h named as FSH primed cells. Subsequently, FSH primed granulosa cells were treated with ovine LH (500 ng/ml) or rhsFRP-4 (0.5 ng/ml or 50 ng/ml) alone or both in combination for further 48h. Conditioned media were harvested after 48h or 96h for estradiol (E2) and progesterone (P4) detection by an ultrasensitive immunoassay enzyme linked assay. Subcellular localization of stabilized known as activated beta-catenin and its colocalization with active caspase-3 was further examined by using mouse monoclonal anti-activated beta-catenin antibody and rabbit polyclonal anti-active caspase-3 antibody which were assessed by double immunofluoresence method.Results: Treatment of cells with rhsFRP4 alone or prior to FSH addition caused a significant increase of P4 secretion. However, using rhsFRP4 in combination with FSH or LH showed less potent effect on E2 or P4 secretion. Moreover, cell treatment prior to LH addition completely inhibit LH-induced P4 secretion. Intersetingly, low dose of rhSFRP-4 strongly induced β catenin stabilization and its nuclear localization which was co-localized with nuclear active caspase-3 as revealed by double immunofluoresence. Conclusion: Our data suggests that low concentration of sFRP-4 could play an agonistic role in Wnt signaling modulation by increasing beta-catenin stabilization and its subsequent nuclear localization which could be associated with apoptosis. Moreover, sFRP-4 could modulates differently granulosa cells FSH- and LH-induced steroidogenesis which may explain the involvement of Wnt signaling pathway in hormonal imbalance and infertility such as polycystic ovary.