PORCINE OVARIAN GRANULOSA-CELLS SECRETE INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN-4 AND PROTEIN-5 AND EXPRESS THEIR MESSENGER RIBONUCLEIC-ACIDS - REGULATION BY FOLLICLE-STIMULATING-HORMONE AND INSULIN-LIKE GROWTH FACTOR-I

Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doubler), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBF-4. These results were compared to those in cultures of immature granulosa cells from 1-3.mm follicles, in which 22-and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22-and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4. The present study demonstrated that IGFBPs of low molecular size are differentially expressed as a function of granulosa cell differentiation and are regulated by hormones and growth factors.