The long term goals of this research are to increase our knowledge of oviductal functions and to understand the complex biochemical and molecular interactions that occur between sperm, ova, and the preimplantation embryo and the maternal oviduct and its secretions that allow fertilization, early cleavage-stage embryonic development and establishment of pregnancy. The goal of this research proposal is: 1) To complete the identification, characterization, distribution and purification of de novo synthesized oviductal secretory proteins (OSP), in particular the estrogen-induced acidic 115,000 Mr and 85,000 Mr proteins; 2) To test for function of OSP; and 3) To study the structure and regulation of OSP by recombinant DNA techniques. We will quantitate production of de novo synthesized OSP throughout the estrous cycle, early pregnancy, in onyx steroid-treated gilts and examine the contribution of the ampulla and isthmus in each and the time span during which they a4e synthesized. We will purify the major OSP (115Kd, 85Kd, pIless than 4) that appear at proestrus and estrus from explant culture medium by ion-exchange, affinity and/or gel filtration chromatography. Production of polyclonal antibodies to the major OSP will allow innunocytochemical detection on oviductal epithelium, sperm, unfertilized ova, and early cleavage-stage embryos and may suggest function. Ultrastructural analysis and histological changes will be correlated with OSP synthesis and distribution. Using in vitro tests we will examine whether OSP have immunosuppressive activity. Further, we will determine whether individual or combined OSP have complement inhibitory activity and can regulate the maternal humoral immune system. We will determine whether sperm, ova, or embryos specifically bind OSP or whether ova and early cleavage-stage embryos take up OSP by endocytosis. In vitro cultures will be used to examine whether the oviduct can stimulate embryonic development. We will purify polyadenylated mRNA for the major OSP (115Kd and 85Kd), examine mRNA translation with and without microsomal membranes, and construct a cDNA library of major mRNA species including those encoding the 115K and proteins. Specific cDNA's will be cloned and characterized, the amino acid sequences derived and protein data base examined for identity. The cDNA's will be used for probes to determine amounts of specific OSP nRNA and to study regulation during the estrous cycle or with hormone treatment. These studies may identify and define mechanisms and controls for fertilization and embryonic development.
