High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro

The present study was aimed to find out whether an increase of cytosolic free calcium level induces egg apoptosis through mitochondria-caspase mediated pathway. To increase cytosolic free calcium level and morphological apoptotic changes, ovulated eggs were cultured in Ca2+/Mg2+ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 mu M) for 3 h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H2O2) concentration, catalase activity, cytochrome c concentration, caspase-9 and caspase-3 activities and DNA fragmentation were analyzed. Calcium ionophore induced morphological apoptotic features in a concentration-dependent manner followed by degeneration at higher concentrations (3 and 4 mu M). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H2O2) and inhibited catalase activity in treated eggs. The increased H2O2 concentration was associated with increased cytochrome c concentration, caspase-9 and caspase-3 activities that resulted in the induction of morphological features characteristic of egg apoptosis. The increased caspase-3 activity finally induced DNA fragmentation as evidenced by TUNEL positive staining in calcium ionophore-treated eggs. These findings suggest that high cytosolic free calcium level induces generation of H2O2 that leads to egg apoptosis through mitochondria-caspase mediated pathway.