Purification and Characterization of Novel Heparin-binding Growth Factors in Uterine Secretory Fluids

Uterine growth factors are potential effector molecules in embryo growth signaling pathways.Pig uterine luminal flushings contained a heparin-binding growth factor (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8.This factor, which was heat-and acid-labile and of M r 10,000 as assessed by gel filtration, stimulated DNA synthesis in fibroblasts and smooth muscle cells but not endothelial cells.Two forms of HBGF-0.8,termed HBGF-0.8-P1 and HBGF-0.8-P2,exhibited differential heparin-binding properties.SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent M r of 10,000 under reducing conditions.Amino acid sequencing revealed the N-terminal sequence EEN-IKKGKKXIRTPKI for HBGF-0.8-P1and ENIKKGKKX-IRT for HBGF-0.8-P2.These sequences corresponded, respectively, to residues 247-262 and 248 -259 of the 349residue predicted primary translation product of porcine connective tissue growth factor (pCTGF). 10-kDa CTGF-mediated fibroblast DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and 20 kDa were present in unfractionated uterine luminal flushings.These data reveal the identity of a novel growth factor in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.Uterine secretions contain a variety of chemical constituents including ions, vitamins, proteins, and carbohydrates that are proposed to sustain the growth and viability of the early embryo during the preimplantation period and, in species that exhibit noninvasive placentation, development of the conceptus after attachment of the extraembryonic membranes to the uter-