The NADPH oxidase 4 is a major source of hydrogen peroxide in human granulosa-lutein and granulosa tumor cells.

HO is a reactive oxygen species (ROS), which can diffuse away from its site of generation and may act as a cell-to-cell signaling factor. The mechanisms responsible for the generation of HO in human ovarian follicles and possible signaling role(s) of HO are not well known. We identified a source of HO, the enzyme NADPH oxidase (NOX) 4, in isolated differentiated, in-vitro fertilisation-derived human granulosa-lutein cells (GCs), in proliferating human granulosa tumour cells (KGN), as well as in situ in cells of growing ovarian follicles. HO was readily detected in the supernatant of cultured GCs and KGN cells. HO levels were significantly lowered by the NOX4 blocker GKT137831, indicating a pronounced contribution of NOX4 to overall HO generation by these cells. We provide evidence that extracellular HO is taken up by GCs, which is facilitated by aquaporins (peroxiporins). We thus conclude that GC-derived HO might act as autocrine/paracrine factor. Addition of HO increased MAPK-phosphorylation in GCs. Moreover, reducing HO production with GKT137831 slowed proliferation of KGN cells. Our results pinpoint NOX4 and HO as physiological players in the regulation of GC functions.