Intracellular tree calcium related to apoptotic cell death in quail granulosa cell sheets kept in serum-free culture

Abstract

The relationship between apoptosis and resting intracellular free calcium ([Ca2+](i)) was studied in serum-free cultures of granulosa cell sheets isolated from preovulatory quail follicles. Apoptosis was detected by acridine orange, in situ end-labeling of fragmented DNA and electron microscopy. [Ca2+](i) was measured using fura-2. [Ca2+](i) averaged 525 mM in freshly isolated sheets. In 24h cultures no apoptosis was detected but [Ca2+](i) became very dispersed, 20% of the sheets showing values above 1000 nM. At 48h, apoptosis was obvious and [Ca2+](i) remained dispersed. At 72 h, apoptosis and also the fraction of sheets with high [Ca2+](i) were at their maximum. At 96h apoptosis was subsiding and [Ca2+](i) normalized. FSH depressed apoptosis and [Ca2+](i) in the 72 h cultures. We conclude that at 24h apoptosis is intitiated at high [Ca2+](i) foci. At later stages apoptosis is associated with high [Ca2+](i), but it is not clear whether this is cause or consequence.