Insulin-Like Growth Factor-Binding Protein-3 in Porcine Ovarian Granulosa Cells: Gene Cloning, Promoter Mapping, and Follicle-Stimulating Hormone Regulation

The role and regulation of IGF-binding protein-3 (IGFBP-3) in the ovary is not fully understood. We cloned and determined the sequence of 12,257 bp of the pig IGFBP-3 gene that includes 4,296 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, whereas the second is a 73-bp region between −63 and +10 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 with 27 additional 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 3 h) and an inhibition at 24 h after 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-I.