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

We have developed a culture system for early bovine embryos in serum-f ree 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 exami ne their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and bioch emical analyses. The cell dissociation procedure yielded two cell popu lations: ciliary cells and secretory cells that gradually dedifferenti ate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and a ll the cells contained cytokeratin filaments as determined by immunocy tochemistry. The overall histoarchitecture of the monolayer was preser ved after washing and further culture for 7 d in serum-free medium. Ho wever, some degenerative signs indicate that the serum-free culture sh ould not be extended for more than 7 d. Confluent oviduct cells also m aintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly i ncreased 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 .