The long term objective of this proposal is to determine how estradiol 17- beta (E2) and progesterone regulate the synthesis and release of an E2- dependent oviduct protein and to directly evaluate its role in reproduction. It is HYPOTHESIZED that this E2-induced protein is synthesized and released into the oviduct lumen during estrus and for the first 3-4 days of pregnancy where it interacts with the gametes to facilitate fertilization and early embryonic cleavage.
Three specific aims are proposed.
SPECIFIC AIM #1. To test the HYPOTHESIS that the synthesis and release of an E2-dependent glycoprotein from the epithelial cells of the ampulla oviduct varies with the estrous cycle and pregnancy so that it is present in the lumen during gamete transport, at the time of fertilization and for the first few days that the developing embryo resides in the oviduct; and that this oviduct protein associates with the oocyte and fertilized ovum. The protein will be immunoprecipitated from oviduct culture media and flushings obtained at select stages in the estrous cycle and early pregnancy and immunocytochemically localized in the oviduct and fertilized egg.
SPECIFIC AIM #2. To test the HYPOTHESIS that this E2-dependent oviduct glycoprotein plays an important role in enhancing in vitro fertilization and embryo development. In vitro fertilization and embryo micromanipulation and transfer techniques will be used to assess the differences in fertilization rates and embryo development in the presence and absence of the E2-dependent protein. Western blot and Northern analysis will be used to assess the viability of in vitro matured embryos by monitoring the production of the embryonic interferon, ovine trophoblast protein-1.
SPECIFIC AIM #3. To test the HYPOTHESIS that this E2-induced oviduct protein is a protein which shares sequence homology with known proteases, enzymes or growth factors. The gene encoding this protein will be cloned and sequenced. The primary amino acid sequence will be inferred from nucleotide sequence and compared for homology to known proteins, particularly proteases, enzymes and growth factors. Collectively, these studies will provide new, direct and important contributions to understand the steroid regulation of the oviduct in early pregnancy.
