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 S-35-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 M(r) = 30 000 and a pl = 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. (125)Insulin-like growth factor-I (IGF-I) 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. AU cells in the junctional zone and the deep glandular region of the implantation site contained IGFBP-1 staining. The synthesis of IGFBP-1 and its release into culture medium appears to be dependent on the process of implantation in the cat and may play an autocrine/paracrine role in the control of trophoblast growth and invasion.