Hormonal control of plasminogen activation in bovine gonadal cells: investigations using domain-specific monoclonal antibodies

Abstract

Two monoclonal antibodies directed to different epitopes of tissue plasminogen activator (tPA), in addition to a chromogenic assay for protease activity, were used to investigate the hormonal control of tPA in bovine Sertoli and granulosa cells. Cells were challenged with a cAMP analogue (8-Bromo-cAMP, 1 mM), a glucocorticoid analogue (dexamethasone, 1 μM) or a combination of both compounds. The protease assay showed that 8-Br-cAMP induced an eight-fold increase in tPA activity over basal levels, and that dexamethasone inhibited this stimulation by 83%. Dexamethasone alone induced a much smaller increase in tPA activity. Similar results were obtained when the active site of tPA was measured in an ELISA using Antibody 3E6, indicating that the proteolytic activity produced by bovine Sertoli cells is largely due to active tPA and can be influenced by both hormones. Bovine granulosa cells also showed a significant (4.7-fold) elevation in tPA activity in response to 8-Br-cAMP, which was inhibited (62%) when dexamethasone was present. Dexamethasone alone resulted in a small (0.9-fold) elevation over basal tPA activity. This profile was also seen when the active site of tPA was measured using Antibody 3E6, and indicates that the mechanisms controlling fibrinolysis are similar in male and female bovine gonadal tissues. When the Antibody WA3 (which binds to the heavy chain of tPA) was used to determine tPA secretion, poor recognition of tPA was seen in all cell supernatants. This lack of binding may be due to masking of the WA3 binding site by fibrin, extracellular matrix proteins, or endogenous activators of tPA, as suggested for rat oocytes.