ABSTRACT. Temporal and spatial distributions of cytokeratin (CK) polypeptides were detected by monoclonal antibodies (mAbs) K8.13 and K8.12 during the development of the bovine ruminal epithelium. By the Western blotting analysis after the sodium dodecyl sulfatepolyacrilamide gel electrophoresis, mAb K8.13 confirmed 60.8 and 63.0 kD CK polypeptides in the fetal ruminal epithelial extract, and mAb K8.12 also 48.0 and 54.0 kD CK polypeptides. Immunohistochemical reactivities against both mAbs were detected only in the epithelial cells throughout the fetal periods. Distributions of CK polypeptides detected only by mAb K8.13 were observed on the basal side of the epitherial layer, but not by mAb K8.12 in the 7 cm fetus in crown-rump length. MAb K8.13 reacted also intensely with columnar-shaped cells in the basal layer in the fetuses of the later developmental periods. These results suggest that CK polypeptides detected by mAb K8.13 might be involved in the differentation and/or the maintenance of the basal layer in the ruminal epithelial development.-KEY WORDS: bovine, cytokeratin, development, epithelium, rumen.J. Vet. Med. Sci. 61(3): 261-265, 1999 ruminal epithelium using two mAbs ; i.e., MAb K8.13 raised against the stratified epithelial type CK polypetides [10,13,14], and mAb K8.12 against the non-stratified hyperproliferative epithelial type CK polypeptides [10,20]. These mAbs recognize the broad-range of CK polypeptides in species including man, cow, chick and amphibia, but show only limited reactivity with only a few rodent CK polypeptides [10].
MATERIAL AND METHODS
Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and Werstern blotting:Fourteen bovine fetuses from 7 to 90 cm in crown-rump length (CRL) were obtained from a local slaughterhouse, Saitama Prefecture, Japan (Table 1). The ruminal epithelium was homogenized in 50 mM Tris-HCl, pH 7.4, with 1 M KCl, 1 mM ethylenediaminetetraacetic acid disodium salt, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreit and 1% Triton X-100 (all reagents were purchased from Sigma Chemical Co., Ltd., U.S.A.), stirred at 4°C overnight, and then centrifugated at 18,000 g for 30 min at 4°C. The precipitate was resolved in the Laemmli's buffer [12] We have been interested in the mechanism of the morphogenetical regulations under the epithelialmesenchymal interaction, and have studied the morphogenesis of the bovine ruminal papillae and the palatine ridge [1,2,[16][17][18]. The epithelial-mesenchymal interaction is known to conduct several morphogenetic processes such as the digestive tract [21] and the chick feather germ [11]. The finger-and/or leaf-like ruminal papillae have regulary aligned on the ruminal inner surface. We have studied temporal and spatial distributions of extracellular matrix such as fibronectin, laminin, chondroitin sulfate proteoglycans and some collagens as the marker of the mesenchymal differentiation, and those of carbonic anhydrase isozyme III as the marker of the epithelial one in the ruminal papillae (RP) [1,16,17] and pa...