Degradation of Extracellular Matrix Proteins by Bovine Embryonic Urokinase-Type Plasminogen Activator

The bovine blastocyst is composed of a one-celled layer of trophectoderm \ud \ud surrounding a fluid filled sac called the blastocoel and a ball of cells called the inner cell \ud \ud mass (ICM). On Day 8 (Day 0=fertilization) in bovine embryos, endodermal cells leave \ud \ud the ICM, migrate over the extracellular matrix (ECM) on the blastocoelic side of the \ud \ud trophectoderm and form a full layer of extraembryonic endoderm on Day 10. Three ECM \ud \ud glycoproteins have been shown to be involved in endodermal cell migration: fibronectin, \ud \ud vitronectin, and laminin. Bovine endodermal cells migrate on fibronectin and vitronectin \ud \ud whereas laminin is believed to be produced when migration is complete to stabilize cell-\ud \ud ECM interactions (Haun and Menino, 2001; Singleton and Menino, 2005). \ud Urokinase-type plasminogen activator (uPA) activity has been detected in bovine \ud endodermal cells during cell migration, but its exact role is not understood (Haun and \ud Menino, 2001). The matrix metalloproteinase (MMP) inhibitor, TIMP-2, is stimulatory to \ud bovine endodermal cell proliferation and migration, and enhances PA production but \ud MMP have not been detected in bovine ICM (Haun and Menino, 2001). Endodermal cell \ud migration may require limited proteolysis of the ECM, and if this is the case uPA may be \ud the protease involved. The objective of this research was to determine if the bovine \ud embryonic uPA can directly degrade fibronectin and laminin without plasminogen \ud activation.\ud Embryos for this project were obtained from cows at the OSU Beef Cattle Center and \ud donations by breeders. Conditioned medium was collected from embryos ranging from \ud morulae (Day 6) to hatched blastocysts (Day 12). Conditioned medium was assayed for \ud embryonic uPA (ePA) activity using a caseinolytic agar gel assay. Proteolysis of the \ud ECM glycoproteins by ePA was determined by SDS-polyacrylamide gel electrophoresis. \ud No autolysis was observed over 48 hr of incubation at 39º C for fibronectin, \ud collagen ІV, or laminin using SDS-PAGE. Lysis was observed for laminin by human \ud urokinase, but no fibronectin breakdown occured with human urokinase. Laminin showed \ud no degradation by bovine ePA, however the polypeptide profile of fibronectin \ud demonstrated cleavage by bovine ePA. These results indicate that ePA can proteolytically \ud degrade fibronectin in the absence of plasminogen. Cleavage of fibronectin may aide in \ud endodermal cell proliferation by facilitating movement over the ECM