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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 24 h cultures no apoptosis was detected but [Ca2+]i became very dispersed, 20% of the sheets showing values above 1000 nM. At 48 h, 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 96 h apoptosis was subsiding and [Ca2+]i normalized. FSH depressed apoptosis and [Ca2+]i in the 72 h cultures. We conclude that at 24 h apoptosis is initiated 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.

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