Identification, characterization and localization of three proteins expressed by the porcine oviduct

Abstract

At estrus, the oviduct undergoes endocrine-induced changes which provide an essential microenvironment for maturation of gametes, fertilization and embryonic development. Several oviduct expressed proteins which interact with gametes or embryos, including the oviduct-specific, estrogen-dependent glycoprotein (OGP), have been identified and characterized. The objective of the present study was to identify, characterize and localize other proteins expressed by the porcine oviduct during estrus that may function in an autocrine or paracrine manner to enhance fertilization and embryonic development. Oviducts were collected during the estrous cycle or early pregnancy, flushed and divided into functional segments, and portions of the infundibulum, ampulla and isthmus were fixed for immunocytochemical analysis or cultured. Culture media was semi-purified by heparin–agarose affinity chromatography, proteins were transferred to polyvinylidene fluoride (PVDF) membrane after two-dimensional (2D)-SDS–PAGE and three different proteins were identified, excised and subjected to N-terminal amino acid analysis. These proteins were identified as complement component C3b, the carboxy-terminal propeptide of alpha 1 (III) procollagen (PIIICP), and the heavy chain variable region of IgA. Electrophoresis and fluorography of media from Days 0 to 12 of early pregnancy or the estrous cycle revealed both spatial and temporal expression of C3b and IgA heavy chain but not PIIICP by the oviduct. Further, all three proteins were identified in oviduct fluid by electrophoresis, immunoblot or immunoprecipitation analysis. Complement component C3b and IgA heavy chain were immunolocalized in all three oviduct segments on all days; however, temporal and spatial differences were demonstrated. Staining was greater in the infundibulum and during estrus for all three identified proteins. In summary, three proteins expressed by the oviduct at estrus and during early pregnancy were identified; characterization and localization suggest they may play a critical role in protecting the luminal environment, participating in ECM remodeling and gamete interactions.