The role and regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12257 bp of the pig IGFBP-3 gene that includes 4296 bp of the flanking promoter sequence. The porcine IGFBP-3 promoter sequence shares two highly conserved regions with the human and bovine IGFBP-3 promoters and a mouse DNA clone. The first is a 38 bp region between -1095 and -1058, while the second is a 73 bp region between -63 and +10

more »

... of the pig sequence. Projected translation of the open reading frame of our sequence gave a peptide sequence identical to that determined by peptide sequencing but adds 27 amino acids upstream of this sequence and is highly similar to the human, bovine, rat and mouse IGFBP-3 peptides. Using RT-PCR we demonstrated that FSH regulates IGFBP-3 mRNA expression in a biphasic manner, with an early induction (maximal at 3h) and an inhibition at 24 h post FSH treatment. The inhibition at 24 h was not due to changes in IGFBP-3 mRNA stability. A similar pattern of FSH modulation of the IGFBP-3 gene transcription was demonstrated by the reporter activity of granulosa cells transiently transfected with IGFBP-3 promoter constructs. The site for FSH stimulation of the IGFBP-3 gene was localized to the sequence between -61 and -48 relative to the transcription start site. Regulation of IGFBP-3 transcription by FSH suggests a role for IGFBP-3 in follicular development that may be independent of IGF-1. response to stimulatory molecules such as TPA, cAMP, GH, IGF-I, FSH, retinoic acid and glucocorticoids. The objectives of this study were to evaluate the mechanism of FSH action on the expression of IGFBP-3 mRNA in cultured porcine ovarian granulosa cells. To accomplish this goal we decided to clone the IGFBP-3 gene, map the promoter sequence and identify putative consensus sites for transcription factors. The last objective was to generate promoter constructs for use in studying FSH and growth factor regulation of the IGFBP-3 gene. Materials and Methods The following reagents were purchased from Sigma Aldrich (St Louis, MO): BioBond R -Plus Nylon Membrane, dNTP Mix, JumpStart Taq DNA polymerase, dichloro-β-Dribofuranosyl-benzimidazole (DRB) and DMSO. We purchased the following supplies from Gibco/Invitrogen Corporation (Grand Island, NY): fetal bovine serum, lipofectin, and gentamicin. Falcon brand cell culture dishes and flasks were purchased from Becton Dickinson LabWare (Lincoln Park, NJ). The reporter lysis buffer, luciferase assay kit and beta galactosidase assay kits were purchased from BD Biosynthesis (Palo Alto, CA).