Endocrine-regulated and protein kinase C-dependent generation of superoxide by rat preovulatory follicles

The ovulatory LH surge results in follicular inflammation with an increase in cytokines and PGs. Reactive oxygen species (ROS) are also produced durin g inflammatory processes. To study ROS generation during the ovulatory casc ade, preovulatory follicles were dissected from immature female rats primed with PMSG. Follicles were isolated, and ROS generation was assessed by lum inol-amplified chemiluminescence. Immature rat granulosa cells were also su bjected to luminometry after isolation from immature rats treated with diet hylstilbestrol. Phorbol ester-stimulated ROS generation by follicular cells was completely suppressed by superoxide dismutase and the NADPH/NADH oxida se inhibitor diphenylene iodonium bisulfate, whereas catalase was without e ffect. Fractionation of granulosa cells with an antibody against leukocyte common antigen-1 showed that leukocyte-enriched cells produced more than 95 % of the superoxide measured. In vivo treatment with LH produced a 5-fold i ncrease in phorbol-stimulated superoxide production by isolated follicles. This response was maximal within 4 h and was blocked by indomethacin. In vi vo administration of PGE(2) and PGF(2 alpha) did not reverse the blockade b y indomethacin; however, isolated follicles incubated with PGE, produced a time-dependent increase in phorbol-stimulated superoxide generation. Thus, a superoxide generator is present in the preovulatory follicle that is leuk ocytic in origin, hormone regulated, and activated by a protein kinase C-de pendent pathway. The regulated generation of superoxide by preovulatory fol licles may indicate a role for ROS in the periovulatory period.