Inhibition of apoptosis in cultured porcine granulosa cells by inhibitors of caspase and serine protease activity

Protease inhibitors were used to test the hypothesis that caspases and othe r proteases were active during apoptosis in cultured porcine granulosa cell s. Cells isolated from 3 to 6 mm follicles were cultured for 24 h in Dulbec co's modified Eagles medium: Hams F12 (1:1') containing 1% fetal bovine ser um. Final inhibitor concentrations, added in 10 muL of dimethylsulfoxide, w ere 0, 1, 5, 25 and 125 muM. Cells with compromised plasma membrane integri ty, identified by uptake ethidium homodimer, increased during culture in th e absence of inhibitors from 37% to 43%.'Apoptotic (A(o)) cells, identified by DNA fluorescence flow cytometry, increased (P < 0.05) from 1.7% to 29%. The serine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) at 125 <mu>M was lethal increasing (P < 0.05) cells with compromised membranes to 92%. In response to TPCK, A(o) cells decreased from 55% to 1. 2%; progesterone and estradiol production were decreased by 94% and 98%, re spectively. The general casp ase inhibitor, benzyloxycarbonyl-valinyl-alani nyl-aspartyl fluoro methylketone, decreased (P < 0.05) A(o) cells linearly from 33% to 3% between 0 and 125 muM without significant effect on steroido genesis or on the percentage of cells with compromised plasma membranes. Ot her inhibitors only had a marginal effect on apoptosis; concentrations of g reater than or equal to1 muM decreased (P < 0.05) A(o) cells from 29% to 18 % to 21% and had no significant effect on membrane integrity or steroid pro duction. We conclude that caspases are associated with apoptosis in culture d porcine granulosa cells. Death induced by TPCK was through a nonapoptotic mechanism. Published by Elsevier Science Inc.