published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 1992.

Thesis (Ph. D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 166-197).

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. / Graduation date: 1993

published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph.D.) - University of Queensland, 1996. / Includes bibliographical references.

Thesis (Ph.D.)--Memorial University of Newfoundland, Faculty of Medicine, 1997. / Typescript. Bibliography: leaves [222]-256.

Abstract not available

Graduation date: 1993

The effects of several hormones and inducers of intracellular messengers on plasminogen activator (PA) production and changes in embryonic size by cultured bovine embryos were evaluated. Day 8 embryos were cultured in Ham's F-12 with 1.5 mg/ml bovine serum albumin (BSA) containing different levels of progesterone (P), estradiol -17fl (E₂), dexamethasone (Dex), retinoic acid (RA), dibutyryl cyclic AMP (dbcAMP), or phorbol myristate acetate (PMA) for 5 days under paraffin oil in a humidified atmosphere of 5% CO₂ in air at 37°C. The concentrations of PA in the conditioned media were determined by a caseinolytic assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymography were used to determine the molecular weight of PA in the medium and in the embryo homogenate. Changes in embryonic size were determined by measuring overall embryo diameter (OD) at 24-h intervals. None of the hormones and agents tested herein had a significant effect on PA production. Dimethyl sulfoxide (DMSO) which was used to dissolve PMA significantly inhibited PA production during the first 72 h of culture. Time of culture, however, exerted a significant effect on PA production by cultured embryos. The production of this protease was low during the first 48 h, increased during 72 and 96 h, and either remained high or slightly decreased toward the end of the culture period. Furthermore, the peak production of PA was attained 48 h after hatching. The molecular weight of PA in the conditioned medium and embryo tissues suggested that the bovine embryo at this developmental stage produced an urokinase-type PA. With the exception of dbcAMP and PMA, the hormones tested in this study did not affect embryonic size. While dbcAMP decreased OD later in culture, PMA enhanced OD throughout culture. The mechanism by which dbcAMP and PMA modulated embryonic size is not clear. These results suggest that cultured bovine embryos produce urokinasetype PA in a time dependent manner and the production of this enzyme is independent of exogenous hormonal regulation. / Graduation date: 1990