H 2 O 2 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 H 2 O 2 in human ovarian follicles and possible signaling role(s) of H 2 O 2 are not well known. We identified a source of H 2 O 2 , 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. H 2 O 2 was readily detected in the supernatant of cultured GCs and KGN cells. H 2 O 2 levels were significantly lowered by the NOX4 blocker GKT137831, indicating a pronounced contribution of NOX4 to overall H 2 O 2 generation by these cells. We provide evidence that extracellular H 2 O 2 is taken up by GCs, which is facilitated by aquaporins (peroxiporins). We thus conclude that GC-derived H 2 O 2 might act as autocrine/paracrine factor. Addition of H 2 O 2 increased MAPK-phosphorylation in GCs. Moreover, reducing H 2 O 2 production with GKT137831 slowed proliferation of KGN cells. Our results pinpoint NOX4 and H 2 O 2 as physiological players in the regulation of GC functions.

Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.