Detection of insulin-like growth factor binding protein-1 in cat implantation sites

This study was undertaken to determine whether insulin-like growth factor binding protein-1 (IGFBP-1) was synthesized by the cat uterus and placenta during implantation and pregnancy. Endometrial and placental tissue explants from pregnant, pseudopregnant, and ovariectomized steroid-treated cats were cultured in the presence of 35S-methionine. Culture media proteins were separated by one-dimensional (1-D) and two-dimensional (2-D) SDS-PAGE, transferred to nitrocellulose, and immunostained using a rabbit polyclonal antibody against baboon IGFBP-1 and a murine monoclonal antibody to human IGFBP-1. The antibody cross-reacted with a protein with an Mr = 30 000 and a pI 5.1-5.4. Immunoreactive product was found in implantation site media from 16 days postcoitum (PC) through the end of pregnancy, and was confined to the superficial placental/junctional zone. Immunoreactivity was not detected in non-implantation site media until 7 wk PC and was never detected in serum or in media from liver, pseudopregnant endometrium, or endometrium from steroid-treated cats. Autoradiography and immunostaining of 2-D Western blots of culture media proteins demonstrated that implantation site and not non-implantation site tissue synthesized and released immunoreactive IGFBP-1 into the culture medium. 125Insulin-like growth factor-1 (IGF-1) specifically bound to this protein on 1-D Western ligand blots. Avidin-biotin immunocytochemistry utilizing the monoclonal antibody was used to localize IGFBP-1 in paraffin sections. Specific immunostaining was observed in the surface and glandular epithelium of the non-site endometrium throughout pregnancy, with stromal cell staining being detected later The placental labyrinth had widespread specific immunostaining, especially in the syncytiotrophoblast and maternal giant cells after 4 wk; after 9 wk, immunostaining could be detected only in the giant cells. All cells in the junctional zone and the deep glandular region of the implantation site conta