NADPH oxidase-generated reactive oxygen species in mature follicles are essential for ovulation.

Ovarian reactive oxygen species (ROS) are believed to regulate ovulation in mammals, but the details of ROS production in follicles and the role of ROS in ovulation in other species remain underexplored. In ovulation, matrix metalloproteinase 2 (MMP2) is required for follicle rupture by degradation of posterior follicle cells surrounding a mature oocyte. We recently demonstrated that MMP2 activation and follicle rupture are regulated by the neuronal hormone octopamine (OA) and the octopamine receptor in mushroom body (OAMB). In the current study, we investigated the role of the superoxide-generating enzyme NADPH oxidase (NOX) in ovulation. We report that is highly enriched in mature follicle cells and that knockdown in these cells leads to a reduction in superoxide and to defective ovulation. Similar to MMP2 activation, NOX enzymatic activity is also controlled by the OA/OAMB-Ca signaling pathway. In addition, we report that extracellular superoxide dismutase 3 (SOD3) is required to convert superoxide to hydrogen peroxide, which acts as the key signaling molecule for follicle rupture, independent of MMP2 activation. Given that homologs are expressed in mammalian follicles, the NOX-dependent hydrogen peroxide signaling pathway that we describe could play a conserved role in regulating ovulation in other species.