Apoptotic granulosa cells have moderately increased intracellular free calcium during phosphatidylserine exposure and a normal resting calcium level during DNA fragmentation

Abstract

Alterations in intracellular free calciumconcentration ([Ca²⁺]_i) areinstrumental in apoptosis. We have previously shown that a[Ca²⁺]_i increase above 1000 nM isrelated to the appearance of apoptosis in serum-free cultures ofgranulosa cell sheets. In the present study we examined how the[Ca²⁺]_i increase relates toindicators of distinct phases of the apoptotic cascade. We used adouble staining technique whereby loading with theCa²⁺ indicator fura-2 and capture of a[Ca²⁺]_i image, was followed bystaining with annexin-V, as an early apoptotic marker or withacridine orange, marking the late degradation phase. Calcium imagingshowed a large heterogeneity of cellular[Ca²⁺]_i levels. [Ca²⁺]_i was moderately increased to∼230 nM in annexin positive cells but was at resting levelin cells with nuclear manifestations of apoptosis as evidenced byacridine orange. Our results suggest that a moderate[Ca²⁺]_i increase is related tophosphatidylserine translocation and that[Ca²⁺]_i has already recovered inapoptotic cells displaying chromatin condensation and/or nuclearfragmentation. Granulosa cells with[Ca²⁺]_i above 1000 nM were neverobserved to stain positive for the apoptotic markers used; therefore,large [Ca²⁺]_i increases areprobably related to the apoptosis initiation phase occurring upstreamof phosphatidylserine exposure.