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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29HD030334-05
Application #
2403303
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Yoshinaga, Koji
Project Start
1993-12-01
Project End
1999-11-30
Budget Start
1997-12-01
Budget End
1999-11-30
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Nationwide Children's Hospital
Department
Type
DUNS #
City
Columbus
State
OH
Country
United States
Zip Code
43205
Uzumcu, M; Homsi, M F; Ball, D K et al. (2000) Localization of connective tissue growth factor in human uterine tissues. Mol Hum Reprod 6:1093-8
Harding, P A; Davis-Fleischer, K M; Crissman-Combs, M A et al. (1999) Induction of anchorage independent growth by heparin-binding EGF-like growth factor (HB-EGF). Growth Factors 17:49-61
Brigstock, D R (1999) The connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed (CCN) family. Endocr Rev 20:189-206
Surveyor, G A; Brigstock, D R (1999) Immunohistochemical localization of connective tissue growth factor (CTGF) in the mouse embryo between days 7.5 and 14.5 of gestation. Growth Factors 17:115-24
Steffen, C L; Ball-Mirth, D K; Harding, P A et al. (1998) Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro. Growth Factors 15:199-213
Ball, D K; Surveyor, G A; Diehl, J R et al. (1998) Characterization of 16- to 20-kilodalton (kDa) connective tissue growth factors (CTGFs) and demonstration of proteolytic activity for 38-kDa CTGF in pig uterine luminal flushings. Biol Reprod 59:828-35
Surveyor, G A; Wilson, A K; Brigstock, D R (1998) Localization of connective tissue growth factor during the period of embryo implantation in the mouse. Biol Reprod 59:1207-13
Harding, P A; Surveyor, G A; Brigstock, D R (1998) Characterization of pig connective tissue growth factor (CTGF) cDNA, mRNA and protein from uterine tissue. DNA Seq 8:385-90
Brigstock, D R; Steffen, C L; Kim, G Y et al. (1997) Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids. Identification as heparin-regulated Mr 10,000 forms of connective tissue growth factor. J Biol Chem 272:20275-82
Brigstock, D R; Kim, G Y; Steffen, C L et al. (1996) High molecular mass forms of epidermal growth factor in pig uterine secretions. J Reprod Fertil 108:313-20

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