FOLLICLE-STIMULATING-HORMONE AND INSULIN-LIKE GROWTH-FACTOR-I ATTENUATE APOPTOSIS IN CULTURED PORCINE GRANULOSA-CELLS

Thymocytes and granulosa cells (GC) were cultured up to 48 h to determ ine the effect of dexamethasone (DEX) on apoptosis in culture (in RPMI containing 10% fetal bovine serum [FBS] at 38 degrees C in a 5% CO2:9 S% air atmosphere), In experiment 2, CC were cultured for 24 h at a de nsity of 0.5 x 10(6) cells/0.5 mi in Dulbecco's Modified Eagle's mediu m:Hams F-12 (1:1) containing 1% FBS to determine whether porcine FSH a nd insulin-like growth factor-I (IGF-I) attenuated apoptosis and to co mpare two methods of measuring apoptosis: 1) Flow cytometry of dispers ed cells for subdiploid DNA Fluorescence and 2) densitometry of intern ucleosomal DNA fragments. The percentages of apoptotic (%A(0)) thymocy tes 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, ap optosis, measured by %A(0) GC and amount of internucleosomal[ fragment s, 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 wer e correlated (r = 0.829, n = 44, p less than or equal to 0.0001), We c onclude that 1) porcine GC and thymocytes undergo spontaneous apoptosi s in culture, 2) two methods of analyzing apoptosis were in excellent agreement, and 3) FSH and IGF-I attenuated spontaneous apoptosis in cu ltured porcine GC.