Research Project
2403303
The broad, long-term objectives of this work are to elucidate the nature and function of uterine secretory growth factors that regulate the growth of the extra-embryonic membranes of the blastocyst and/or proliferation of endometrial epithelium or stroma. This proposal focusses on the characterization of a novel growth factor in pig uterine luminal flushing that is elevated in early pregnancy. This factor, a cationic, 10,000-Mr, heat-labile, acid-labile mitogen for fibroblasts, smooth muscle and endometrial cells, has been termed "heparin-binding growth factors. The specific aims of this project are 1) TO isolate and molecularly characterize HBGF-0.8; 2) To analyze the nature and functional significance of heparin-HBGF-0.8 interactions; 3) To quantify HBGF-0.8 in uterine fluids; and 4) To study HBGF-0.8 receptor localization and function in the uterine tract. The health relatedness of this project is that HBGF-0.8 may (i) promote blastocyst development and reduce the incidence of embryonic mortality in utero or after in vitro fertilization and (ii) stimulate cyclic endometrial remodelling or contribute to the onset and progression of uterine tract cancers. The research design and methods are to purify HBGF-0.8 to homogeneity using preparative column chromatography (e.g. cation exchange, heparin-affinity, reverse-phase) and to determine this amino acid sequence directly or indirectly by cloning and sequencing its cDNA. Purified HBGF-0.8 will be labelled with I to study (i) its binding to heparin-like molecules in extracellular matrix and cell surfaces (ii) the nature of its specific high affinity receptors and (iii) the presence of functional HBGF-0.8 receptors on freshly isolated or cultured endometrial or trophoderm cells. The effect of heparin on mediating HBGF-0.8 will be mapped by determining which synthetic HBGF-0.8 peptides bind to heparin and modulate binding of HBGF- 0.8 to heparin. Rabbits will be immunized with synthetic peptides that are conjugated to a promiscuous tetanus toxoid T-cell epitope. Specific anti-HBGF-0.8 antibodies will be selected by ELISA. Antisera will be used to screen recombinant gammagt11 for HBGF-0.8, to immunolocalize HBGF-0.8 in uterine sections, to quantify HBGF-0.8 competitive ELISA in uterine flushing from estrous, pregnant, or steroid-treated pigs, and for detecting HBGF-0.8 on Western blots.
