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