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DIFFERENTIATIVE EFFECTS OF IGF SYSTEM COMPONENTS ON BOVINE FOLLICULAR CELLS.
Nicholas, Ben1, Webb, Robert1, Hogg, Charis2, Baxter, Gerry2, Goddard, Chris3, Armstrong, David2, 1 2 3
ABSTRACT- The insulin-like growth factors are know to have multiple effects on a range of cell types, including an enhancement of cell proliferation, altered susceptibility to apoptosis, changes in cell morphology, and differentiation. The bioavailibility of IGFs is controlled by binding to the IGF binding proteins (IGFBPs), which sequester free IGF's and can either inhibit or enhance their biological effects. IGFBP's -2 and -5 are known to bind to components of the extracellular matrix, as well as directly to cells, and previous studies have identified IGFBP-2 as a key factor regulating IGF bio-availibility in bovine follicles, however there is limited information available about the effect of IGFBPs on follicular cell differentiation. In this study we describe the effects of IGFBP-2 on bovine granulosa cells in-vitro, and its' binding to various components of the extracellular matrix (ECM). We have examined the binding of biotinylated recombinant bovine IGFBP-2 to purified ECMs in a microtitre plate assay, and to the ECM of cells cultured on surfaces coated with these matrices. IGFBP-2 showed greatest affinitiy for fibronectin of the matrices tested (compared to collagen IV, laminin, purified ECM, poly-l-lysine, fibronectin-like engineered polymer or uncoated surfaces), however when granulosa cells were cultured on matrix-coated surfaces, exogenous IGFBP-2 bound least to the ECM of cells cultured on fibronectin coated plates. IGFBP-2 binding to cellular ECM was significantly reduced in a dose-dependent manner upon treatment with FSH (0,1,5 and 10 ng/ml), indicating either a reduction in ECM production by these cells or an alteration in ECM biochemistry. Estradiol production was stimulated by FSH when the cells were cultured on fibronectin, collagen or purified ECM, whereas progesterone secretion was increased on all surfaces compared to uncoated controls. Although gross cellular morphology was altered when cells were grown on different ECMs, the assumption of a flattened morphology was not always associated with the loss of estradiol production, as had been expected. These results demonstrate that IGFBP-2 can interact with components of the ECM in a gonadotropin-dependent manner. This may be an important mechanism whereby granulosa cells can regulate their exposure to IGFs. The work was funded by the BBSRC.
KEY WORDS: IGF, ECM, Differentiation, IGFBP