Electrophoretic characterization of the plasminogen activator produced by bovine blastocysts.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and zymography were used to determine the tissue source and to characterize the types of plasminogen activator (PA) produced by bovine blastocysts. Day 12-14 blastocysts were collected at slaughter from oestrus-synchronized, superovulated and artificially inseminated Holstein cows. In Expt 1, blastocysts were cultured for 24 h in Ham's F-12 in a humidified atmosphere of 5% CO2 in air at 37 degrees C. After culture, blastocysts and medium were recovered and stored separately at -20 degrees C. In Expt 2, embryonic discs were separated from trophoblast by microdissection. Intact blastocysts, embryonic discs and trophoblast were then cultured for 24 h and recovered as in Expt 1. In both experiments, embryonic tissues and media were electrophoresed with PA and molecular mass standards. Polyacrylamide gels were laid onto casein-agar gel plates (zymograms) and incubated at room temperature for 24-48 h. Caseinolytic zones in zymograms containing plasminogen were evidence of PA. In Expt 1, bovine blastocysts contained and secreted light and heavy forms of PA (47.0 +/- 1.0 and 86.1 +/- 0.7 kDa, respectively). In Expt 2, intact blastocysts and trophoblast produced both forms of PA (41.5 +/- 1.5 and 92.2 +/- 2.7 kDa) but PAs were not detected in embryonic discs. The results suggest that Day 12-14 bovine blastocysts produce urokinase-type PA (41.5-47.0 kDa) and a form of high molecular mass (86.1-92.2 kDa) that is either a novel tissue-type PA or a PA inhibitor which complexes with the lighter form.