HIGH AND LOW-MOLECULAR-WEIGHT DNA CLEAVAGE IN OVARIAN GRANULOSA-CELLS- CHARACTERIZATION AND PROTEASE MODULATION IN INTACT-CELLS AND IN CELL-FREE NUCLEAR AUTODIGESTION ASSAYS

To continue elucidation of the biochemical and molecular pathways invo lved in the induction of apoptosis in granulosa cells (GC) of ovarian follicles destined for atresia, we characterized the occurrence and pr otease modulation of high and low molecular weight (MW) DNA fragmentat ion during rat GC death, Atresia of ovarian follicles, occurring eithe r spontaneously in vivo or induced in vitro, was associated with both high MW and internucleosomal (low MW) DNA cleavage, Incubation of foll icles in the presence of a putative irreversible and non-competitive i nhibitor of caspase-1 (interleukin-1 beta-converting enzyme or ICE), s odium aurothiomalate (SAM), completely prevented internucleosomal, but not high MW, DNA cleavage, As reported previously, morphological feat ures of apoptosis (pyknosis, cellular condensation) and atresia (granu losa cell disorganization, oocyte pseudomaturation) remained detectabl e in SAM-treated follicles, The potential involvement of proteases in endonuclease activation was further analyzed in cell-free assays using nuclei from both GC (which autodigest their DNA) and HeLa cells (HC, which do not autodigest their DNA un less incubated with extracts prep ared from other cell types). Crude cytoplasmic extracts prepared from GC induced both high MW and internucleosomal DNA cleavage in HC nuclei . The induction of low, but not high, MW DNA cleavage in HC nuclei by GC extracts was suppressed by pretreatment of the extracts with SAM or with any one of the serine protease inhibitors, dichloroisocoumarin ( DCI), N-tosyl-L-leucylchloromethylketone (TLCK) or N-tosyl-L-phenylchl oromethylketone (TPCK). Interestingly, SAM and DCI also prevented cati on-induced low MW DNA fragmentation in GC nuclei; however, TLCK and TP CK were without effect. Our results support a role for cytoplasmic and nuclear serine proteases in the activation of the endonuclease(s) res ponsible for internucleosomal DNA cleavage during apoptosis.