Graduate Thesis Or Dissertation
 

Factors affecting plasminogen activator activity in bovine and porcine oocyte-cumulus cell complexes matured in vitro

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  • Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), okadaic acid (OA), cycloheximide (CHX), actinomycin D (AcD) and tunicamycin (TuM) on plasminogen activator (PA) activity and maturation rate in bovine and porcine oocyte-cumulus cell complexes (BOCC and POCC, respectively) in vitro were determined. Plasminogen activator activity was measured by SDS-PAGE, casein-agar zymography and densitometry. Three plasmingen-dependent lytic zones (92-95, 71-73 and 49-51 kD) and one PA inhibitor (52 kD) were observed in BOCC. Immunoprecipitation and amiloride sensitivity suggested that the 49-51 kD protease is a urokinase type PA (uPA), the 71-73 and 92-95 kD proteases are a tissue type PA (tPA) and tPA-PAI complex, respectively, and the PAI is PAI-1. In POCC, two plasminogen activators (71-78 and 93-96 KD) were observed. Lack of amiloride sensitivity suggested that the 71 -78 kD band is a tPA and the 93-96 kD band is possibly a tPA-PAI complex. Increasing dbcAMP in the culture medium increased activity in both BOCC and POCC in dose-dependent fashion (P<0.05). In BOCC cultured with PMA, total PA activity increased, however high concentrations of PMA (10 and 100 ng/ml) decreased tPA activity in matured POCC. Plasminogen activator activity decreased in 6-DMAP, actinomycin D and cycloheximide and oocyte maturation was also inhibited in these treatments. When POCC were treated with 25 nM OA, uPA activity was observed. Plasminogen activator activity increased in either BOCC or POCC treated with up to 25 nM OA, however PA activity decreased at concentrations greater than 75 nM (P<0.05). Incubation of BOCC with tunicamycin reduced the molecular mass of tPA and tPA-PAI complex and PAI-1 by 5-10%, however PA activity was not inhibited. These data suggest that BOCC matured in vitro produce uPA, tPA and PAI-1 however POCC produce only tPA and PAI. The production of PA and PAI by either BOCC or POCC is associated with oocyte maturation and influenced by stimulators of the protein kinase A and C, modulators of intracellular phosphorylation and metabolic inhibitors.
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