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

Abstract

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 Ca(2+)/Mg(2+) free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4μM) for 3h in vitro. The morphological apoptotic changes, cytosolic free calcium level, hydrogen peroxide (H(2)O(2)) 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 4μM). Calcium ionophore increased cytosolic free calcium level, induced generation of hydrogen peroxide (H(2)O(2)) and inhibited catalase activity in treated eggs. The increased H(2)O(2) 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 H(2)O(2) that leads to egg apoptosis through mitochondria-caspase mediated pathway.