Follicle-stimulating hormone and insulin-like growth factor-1 attenuate apoptosis in cultured porcine granulosa cells

Thymocytes and granulosa cells (GC) were cultured up to 48 h to determine the effect of dexamethasone (DEX) on apoptosis in culture (in RPMI containing 10% fetal bovine serum [FBS] at 38 degrees C in a 5% CO2:9S% air atmosphere), In experiment 2, CC were cultured for 24 h at a density of 0.5 x 10(6) cells/0.5 mi in Dulbecco's Modified Eagle's medium:Hams F-12 (1:1) containing 1% FBS to determine whether porcine FSH and insulin-like growth factor-I (IGF-I) attenuated apoptosis and to compare two methods of measuring apoptosis: 1) Flow cytometry of dispersed cells for subdiploid DNA Fluorescence and 2) densitometry of internucleosomal DNA fragments. The percentages of apoptotic (%A(0)) thymocytes and GC increased (p < 0.01) during 48 h of culture, Compared to no DEX, 0.1 or 1.0 mu M DEX in thymocytes caused a 33% further increase (p < 0.01) in %A(0) cells but had no effect in GC, In experiment 2, apoptosis, measured by %A(0) GC and amount of internucleosomal[ fragments, decreased (p < 0.01) by 50% during culture in the presence of FSH (4 NIH-S1 mU/ml) or IGF-I (50 ng/ml); results from these techniques were correlated (r = 0.829, n = 44, p less than or equal to 0.0001), We conclude that 1) porcine GC and thymocytes undergo spontaneous apoptosis in culture, 2) two methods of analyzing apoptosis were in excellent agreement, and 3) FSH and IGF-I attenuated spontaneous apoptosis in cultured porcine GC.