REGULATION OF INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEIN PRODUCTION BY HUMAN LUTEINIZING GRANULOSA-CELLS CULTURED IN DEFINED MEDIUM

The production of insulin-like growth factor (IGF) binding proteins (IGFBPs) by human luteinizing granulosa cells obtained at oocyte harvest for in vitro fertilization was studied in defined medium. Three species of IGFBP were noted on Western ligand blotting of conditioned medium. A 31-33 kilodalton (kDa) doublet identified as IGFBP-2 by immunoprecipitation was consistently seen and was shown by metabolic labeling to be synthesized de novo. IGFBP-2 production was inhibited by hCG (100 ng/mL) to 32 +/- 8% of levels in control cultures; a similar inhibition was seen with dibutyryl cAMP and forskolin, but not with FSH. IGFBP-2 production was also inhibited by IGF-II and [Leu27]IGF-II (ED50 for both 3 ng/mL) but not by IGF-I at up to 30 ng/mL. In addition to IGFBP-2, a 35-45 kDa IGFBP identified by immunoprecipitation as IGFBP-3 was variably present on ligand blots of control conditioned medium and was consistently found in medium from cells treated with IGF-I or IGF-II (ED50 3 ng/mL) but not with insulin at 2 mg/mL. No de novo synthesis of IGFBP-3 was noted, suggesting that IGFBP-3 may be released from the cell surface by ligand. A 24-kDa IGFBP consistent with IGFBP-4 was also variably noted in basal cultures. Since IGFBPs are believed to sequester IGF peptides and inhibit their action on the granulosa, control of IGFBP production may be an important step in regulating gonadotropin and IGF action in the human granulosa cell.