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Electrophoretic characterization of plasminogen activators produced by early bovine embryos

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  • Sodium dodecyl sulfate polyacrylamide gel electrophoresis and zymography were used in two separate experiments to determine the tissue source and type of plasminogen activator (PA) produced by bovine blastocysts. Twelve-14 d blastocysts were collected at slaughter from estrous synchronized, superovulated and artificially inseminated Holstein cows. In Experiment I, blastocysts were cultured for 24 h in Ham's F- 12 with 15 mg /ml bovine serum albumin under paraffin oil in a humidified atmosphere of 5% CO₂ in air at 37°C. Following culture, blastocysts and media were recovered and stored separately at -20°C until further analysis. In Experiment II, embryonic discs were separated from trophoblast by microdissection, and undissected blastocysts, embryonic discs and trophoblast were cultured for 24 h, recovered and stored as in Experiment I. In both experiments, embryonic tissues and media were thawed and co-electrophoresed with urokinase standards and molecular weight markers. Polyacrylamide gels were laid onto casein-agar gel plates (zymography) and incubated at room temperature for 24-48 h. Lytic zones in casein-agar gels containing human plasminogen were evidence of the presence of PA. In Experiment I, bovine blastocysts contained and secreted both urokinase-type (47.0 ± 1.0 kD) and tissue-type PA (86.1 ± 0.7 kD). In Experiment II, undissected blastocysts and trophoblast produced both urokinase-type (41.5 ± 1.5 kD) and tissue-type PA (92.2 ± 2.7 kD). Plasminogen activators were not detected in embryonic disc tissue or the respective culture medium. The results suggest that 12- 14 d bovine blastocysts produce both urokinase-type and tissue-type PA, and the tissue source of PA is the trophoblast.
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