Characterization of insulin-like growth factor binding proteins secreted by cultured bovine theca and granulosa cells

Abstract

Insulin-like growth factor binding proteins (IGFBPs) secreted by bovine granulosa and theca interna cells cultured in the presence of different luteinizing factors — insulin (2 μg/ml), forskolin (10 μM), or a combination of the two were examined and characterized. Direct binding of [ 125I]IGF to the conditioned media was compared to progesterone production under these different treatments. In theca cells, maximal secretion of IGFBPs was achieved using forskolin alone, whereas maximal progesterone production was induced by the insulin + forskolin treatment. In contrast, maximal secretion of both IGFBPs and progesterone in granulosa cells was achieved using forskolin alone. IGFBP species secreted by the two cell types under the different treatments were detected by ligand blotting. Conditioned media from theca cells in serum-free medium collected on the seventh day of culture exhibited three bands of 34, 40 and 44 kDa when treated with insulin or forskolin. The intensity of the 40–44 kDa complex was enhanced and a 21 kDa band appeared when cells were treated with a combination of insulin plus forskolin. Conditioned media of granulosa cells stimulated with insulin or forskolin exhibited 21, 27, 29, 34 and 40–44 kDa bands. Treatment with insulin + forskolin greatly increased the intensity of a 40–44 kDa complex. A similar shift towards high molecular weight binding proteins was observed when these media were analyzed by high-performance liquid chromatography gel filtration. These findings substantiate the secretion of IGFBPs by bovine theca and granulosa cells and show it to be dependent on culture treatment. The positive correlation between the secretion of a 40–44 kDa complex (possibly IGFBP-3) and cell luteinization indicates the former's importance in ovarian function.