PARTIAL CHARACTERIZATION OF THE PLASMINOGEN-ACTIVATOR PRODUCED BY OVINE EMBRYOS IN-VITRO

The type of plasminogen activator (PA) produced by Day 7, 9, and 11 ovine embryos in vitro was partially characterized. PA activity in the conditioned medium did not change (p > 0.05) up to equivalent gestational Day (EGD) 14 but increased to peak levels on EGD 15 (p < 0.05). Zymographic analysis of conditioned medium and embryos revealed one plasminogen-dependent lytic zone (48-51 kDa) within EGD 8-15 and a second plasminogen-dependent lytic zone that appeared after EGD 13 (79-83 kDa). Addition of the urokinase competitive inhibitor, amiloride, to the zymograph inhibited plasminogen-dependent lytic activity; and forms of both high and low molecular mass were immunoprecipitated with antiserum to bovine urokinase-type PA. PA activity in conditioned medium was completely suppressed by amiloride treatment (p < 0.05) but was increased (p < 0.05) after incubation with antibodies to human PA inhibitor (PAI)-2. Treatment with antibodies to human PAI-I did not increase (p > 0.05) PA activity in conditioned medium. These results suggest that Day 7, 9, and 11 ovine embryos produce a urokinase-type PA (49.9 +/- 0.5 kDa) and possibly a PAI-2-like protein that associates with the PA and forms a PA-PAI complex of high molecular mass (81.4 +/- 1.2 kDa).