Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes

Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.