Follicle-Stimulating Hormone and Insulin-Like Growth Factor-I Attenuate Apoptosis in Cultured Porcine Granulosa Cells1

Abstract

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% CO2:95% air atmosphere). In experiment 2, GC were cultured for 24 h at a density of 0.5 x 10(6) cells/0.5 ml 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 (%A0) thymocytes and GC increased (p < 0.01) during 48 h of culture. Compared to no DEX, 0.1 or 1.0 microM DEX in thymocytes caused a 33% further increase (p < 0.01) in %A0 cells but had no effect in GC. In experiment 2, apoptosis, measured by %A0 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 < or = 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.