Long-term objectives: A better understanding of the role of uterine secretions could lead to methods to 1) reduce post-fertilization pregnancy failure and 2) control the rate of fetal growth. Objectives are to understand the functional and biochemical characteristics of the uterine milk proteins (UTMP), the major proteins secreted by the endometrium of the sheep uterus during pregnancy. Preliminary findings that UTMP inhibit T lymphocyte function in vitro and bind to IgA suggest they may protect the fetus from immunological attack by the mother.
Specific aims and methods: (1) Characterize the binding of IgA to UTMP. Binding of 125-IgA to UTMP conjugated to Sepharose will be determined in several experiments. Binding data will be analyzed by Scatchard Analysis to determine the affinity constant. Binding of 125I-IgA to UTMP-Seph in the presence of IgG, IgM and other proteins will be evaluated to determine specificity of the binding reaction. Proteolytic fragments of 125I-IgA will be tested for binding to UTMP-Seph to determine if UTMP binds to the Fc or F(ab)2 portion of IgA. To verify that binding occurs in utero, uterine fluid will be fractionated by gel filtration and analyzed by immunological procedures for the presence of IgA-UTMP complexes. (2) Test whether binding of UTMP to IgA inhibits biological functions of IgA. UTMP will be tested for their ability to inhibit binding of 125I-IgA to Fc receptors on bacterial and lymphocyte surfaces. If UTMP are found to bind to the F(ab)2 portion of IgA, the ability of UTMP to inhibit binding of specific IgA antibodies to antigen will be evaluated. (3) Determine whether UTMP are similar proteins to secretory component by evaluation of immunological cross-reactivity using immunodiffusion techniques. (4) Test whether UTMP have immunosuppressive activity in utero by determining whether infusion of UTMP in utero prevents rejection of intrauterine skin grafts. (5) Determine the types of oligosaccharide chains on the two forms of UTMP by gel filtration, glycopeptide mapping, lectin binding and gas chromatography. (6) Determine if the formation of the two UTMP from a common, lower molecular weight precursor is due to glycosylation of the precursor. The precursor and the two UTMP will be immunoprecipitated from endometrial tissue treated with a glycosylation inhibitor. The precipitates will be analyzed by SDS electrophoresis to determine if the formation of the two UTMP from the precursor occurs in the absence of glycosylation.
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