Effects of stimulators of protein kinases A and C and modulators of phosphorylation on plasminogen activator activity in porcine oocyte-cumulus cell complexes during in vitro maturation.

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte-cumulus cell complexes (POCC) in vitro were determined. Cumulus cell-enclosed oocytes were collected from 1-4 mm antral follicles and cultured in TCM-199 with 0.3% polyvinylpyrrolidone for 48 hr. PA activities in POCC were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Two plasminogen-dependent lytic zones (93-96 kD and 71-79 kD) were observed in POCC. Addition of amiloride to the zymography, a competitive inhibitor of urokinase-type PA, failed to reduce activities in either zone, suggesting that the 71-79 kD band is a tissue-type PA (tPA) and the 93-96 kD band is possibly a tPA-inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P < 0.05) activity in dose-dependent fashion, whereas 6-DMAP and 10 and 100 ng/ml PMA inhibited (P < 0.05) PA activity. PA activity increased (P < 0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P < 0.05) at concentrations > 75 nM. Treatment with 25 nM OA also induced the expression of an amiloride-sensitive PA (49-52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P < 0.05) by 2.5 mM dbcAMP and 2 mM 6-DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P < 0.05) only progression to metaphase II.(ABSTRACT TRUNCATED AT 250 WORDS)