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 Ca(2+)/Mg(2+) 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 (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 4 μM). 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.
Cell Physiology Laboratory, Biochemistry Unit, Department of Zoology, Banaras Hindu University, Uttar Pradesh, India.
Anima
A
Tripathi
Shail K
SK
Chaube
D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D061207:Calcium Ionophores / Q000494:pharmacology; D020013:Calcium Signaling; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D048430:Cell Shape / Q000187:drug effects; D048429:Cell Size; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism*; D053938:DNA Fragmentation; D005260:Female; D006861:Hydrogen Peroxide / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D051381:Rats
D061207:Calcium Ionophores; D045304:Cytochromes c; D006861:Hydrogen Peroxide; D002374:Catalase; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D002118:Calcium