High cytosolic free calcium level signals apoptosis through mitochondria-caspase mediated pathway in rat eggs cultured in vitro |
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Authors: | Anima Tripathi Shail K Chaube |
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Institution: | (1) Cell Physiology Laboratory, Biochemistry Unit, Department of Zoology, Banaras Hindu University, Varanasi, 221 005, Uttar Pradesh, India; |
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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 Ca2+/Mg2+ free media-199 with or without various concentrations of calcium ionophore (0.5, 1, 2, 3, 4 μM) 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 μM). 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. |
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