HYDROGEN-PEROXIDE SUPPRESSES LOW-DENSITY-LIPOPROTEIN (LDL) UPTAKE AND LDL-SUPPORTED STEROIDOGENESIS BY PORCINE LUTEAL CELLS

The present study tested the hypothesis that hydrogen peroxide (H2O2) inhibits low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by luteal cells. LDL uptake: dispersed porcine luteal cells from mid-cycle (days 6-11, estrus = day 0) were incubated for 0-120 min at 37 degrees C in F-10 medium + 0.1% BSA containing various concentrations of H2O2 (0-1000 mu M). Cells were washed with catalase (2800 U/ml), and then with fresh medium. Cell viability based on trypan blue exclusion was unaltered by H2O2 exposure through 60 min. H2O2-exposed cells were incubated with fluorescent-tagged-LDL (Dil-LDL; 1 mu g/ml) for 10 min at 37 degrees C. Fluorescence of small (SLC) and large (LLC) luteal cells was analyzed by flow cytometry (n = 6 experiments). H2O2 (greater than or equal to 10 mu M) caused a progressive reduction (P < 0.01) in mean fluorescence intensity (MFI) of SLC and LLC indicative of up to a 30-35% decline in LDL uptake. Progesterone (P) production: dispersed luteal cells (4 x 10(4)/0.2 ml) were pre-cultured in DMEM/F-12 medium overnight (similar to 18 h) in 96-well culture plates. Wells were rinsed and fresh media (0.2 ml) containing H2O2 (0-500 mu M) was added. After 30 min, the following treatments were added: human(h)LDL (0 or 50 mu g/ml), human chorionic gonadotropin (hCG; 0 or 100 ng/ml), hCG + LDL, or 22R-hydroxycholesterol (22[OH]-C; 0 or 25 mu g/ml). Cells were incubated for an additional 4 h, and P concentrations in final media samples were measured by RIA. P production was increased (P < 0.05) above basal levels by hCG (1.7x), LDL (2.2x), hCG + LDL (2.9x), and 22(OH)-C (2.4x). H2O2 (10-500 mu uM) dose-dependently suppressed (P < 0.05) P production in all treatment groups. The relative sensitivities of treatments to H2O2 inhibition on the basis of calculated ED(50) (effective doses yielding 50% inhibition) differed as follows: hCG (ED(50) = 72 mu M)approximate to LDL (ED(50)=80 mu M) approximate to hCG+LDL (ED(50) = 110 mu M) > basal (ED(50) > 500 mu M)> 22(OH)-C (ED(50) > 5000 mu M). LDL endocytosis/metabolism may be an important target of oxygen radical attack during functional luteal regression.