CHARACTERIZATION OF BOVINE OVIDUCT EPITHELIAL CELL MONOLAYERS CULTURED UNDER SERUM-FREE CONDITIONS

摘要

We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes.The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analysesThe cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplementedwith 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemi stiy. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in strum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d Confluent oviduct cells also maintainedtheir metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.