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 during inflammatory processes. To study ROS generation during the ovulatory cascade, preovulatory follicles were dissected from immature female rats primed with PMSG. Follicles were isolated, and ROS generation was assessed by luminol-amplified chemiluminescence. Immature rat granulosa cells were also subjected to luminometry after isolation from immature rats treated with diethylstilbestrol. Phorbol ester-stimulated ROS generation by follicular cells was completely suppressed by superoxide dismutase and the NADPH/NADH oxidase inhibitor diphenylene iodonium bisulfate, whereas catalase was without effect. 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 increase in phorbol-stimulated superoxide production by isolated follicles. This response was maximal within 4 h and was blocked by indomethacin. In vivo administration of PGE2 and PGF2a did not reverse the blockade by indomethacin; however, isolated follicles incubated with PGE2 produced a time-dependent increase in phorbol-stimulated superoxide generation. Thus, a superoxide generator is present in the preovulatory follicle that is leukocytic in origin, hormone regulated, and activated by a protein kinase C-dependent pathway. The regulated generation of superoxide by preovulatory follicles may indicate a role for ROS in the periovulatory period. (Endocrinology 142: 687–693, 2001) T OVULATORY SURGE of LH results in follicular hyperemia, edema, vasodilatation, and extravasation of leukocytes, all of which are characteristic of an inflammatory response (1). Various inflammatory mediators play a role in ovulation, including cytokines (2–4), peptides (5), PGs (6), and other phospholipid-derived compounds, such as platelet-activating factor (7–9). The importance of PGs for ovulation was first demonstrated by blockade of ovulation with cyclooxygenase inhibitors (10, 11), and similar studies have subsequently shown that antihistamines (12), antagonists of platelet-activating factor (7–9) and lipoxygenase (13), and antibodies directed against interleukin-8 (IL-8) (4) are also able to block or at least suppress the ovulatory rate. At the time of ovulation, follicular macrophages increase by 5-fold, whereas neutrophils increase by 8-fold (14), and this may be influenced by the local production of various chemotactic cytokines, including IL-1, tumor necrosis factor-a, IL-8, monocyte chemotactic protein-1, and granulocyte monocyte colony stimulating factor (2, 15, 16). The significance of ovarian leukocytes is underscored by the finding that infusion of leukocytes into the perfused rat ovary produced an increased ovulatory rate compared with infusion of LH alone (17), and subsequent studies by Brannstrom et al. (18) demonstrated that IL-1 and tumor necrosis factor-a also increased the ovulation rate in this model. Therefore, in addition to their traditional roles in scavenging cell debris, antigen processing, and promoting repair, leukocytes appear to enhance the physiological processes of follicle growth and ovulation (19). One important consequence of leukocyte infiltration is the generation of reactive oxygen species (ROS) that are associated with acute inflammation. There is indirect evidence that ROS play a role in ovulation; for example, the expression of superoxide dismutase isozymes (Mn-SOD and Cu/Zn-SOD) varies during the periovulatory period (20, 21), and Mn-SOD activity decreases during this time (21). Furthermore, inactivation of superoxide (SO) by long-acting SOD administration blocks ovulation (21, 22). Yet, there is no information regarding the generation of SO during the periovulatory period and its source. Therefore, the objectives of the present studies were to examine the nature and regulation of ROS production by the preovulatory follicle in response to LH and other mediators of ovulation. Materials and Methods