Granulosa-thecal cell interactions in the regulation of plasminogen activator activity during ovarian follicular development in the hen

In the present studies, hen granulosa and thecal cells from the first (F1) and fourth (F4) largest and developing large white follicles (LWF) were cultured alone or cocultured, and plasminogen activator (PA) activity was determined. The PA of the cells (PAc) and the medium (PAm) was measured through use of the chromogenic substrate Val-Leu-Lys-p-nitroanilide, and the total PA (PAt) was calculated. The PA activity of cultured granulosa cells from all stages of follicular development increased with time of culture. Granulosa cell PAc and PAm activity differed with follicular development: the LWF granulosa cells had the highest levels of activity, the F4 had intermediate levels, and the F1 cells had the lowest. Thecal cell PA activity increased during culture but was unaffected by the stage of follicular development. Cocultures of granulosa and thecal cells from F1 follicles exhibited PA activity 2- to 3-fold higher than the sum of the activities of granulosa and thecal cells cultured alone. The PA activity of granulosa cells from LWF was not affected by coculture with thecal cells. Conditioned medium from thecal cells (TCCM) of all stages of follicular development stimulated PAc activity of granulosa cells from F1 and F4 follicles. Conditioned medium from thecal cells of F4 and LWF caused small inhibitory effects on the PAc activity of granulosa cells from LWF. Zymographic analysis of the PA activity of F1 granulosa cell cultures indicates that the enzyme activity is associated with a molecular mass of about 33 kDa, which is consistent with that of urokinase type PA. Thecal cell PA activity was unaffected by granulosa cell-conditioned medium. The factor(s) in the TCCM that stimulated granulosa cell PA activity was thermolabile and sensitive to trypsin digestion but was partially destroyed by acidification. However, the TCCM factor(s) was not extractable with diethyl ether or adsorbed with charcoal:dextran. The effect of TCCM was mimicked by phorbol 12-myristate 13-acetate (PMA; 4-200 ng/ml), which increased PAc activity in F1 and granulosa cells while slightly inhibiting the PAc activity in granulosa cells from LWF. This study demonstrates the interactions between granulosa and thecal cells at late, but not early, stages of follicular development in the regulation of the follicular PA system.