Concentration dependent antioxidative and apoptotic effects of sulforaphane on bovine granulosa cells in vitro

•2–10 μM of SFN had no adverse effects on GC proliferation, whereas 20 μM restrict growth and proliferation.•Treatment with SFN results higher expression of NRF2 and its downstream antioxidant genes in GCs.•At high concentrations SFN also increases the expression of genes related to apoptosis.•ROS and lipid droplets are found to be higher at higher SFN concentrations.•Higher mitochondrial activity was observed in lower concentrations and vice versa.

Sulforaphane (SFN) has received a great deal of research attention because of its ability to induce the production of a battery of antioxidant enzymes in certain concentrations through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which may effectively neutralize reactive oxygen species (ROS) induced oxidative stress. This study was conducted to investigate the potential of different concentrations of SFN in inducing antioxidative and apoptotic effects in granulosa cells (GCs). For this purpose, bovine GCs were collected from preovulatory antral follicles and cultured with different concentrations of SFN (0–80 μM) and based on phenotypic evaluation three concentrations were selected: 2 μM (low), 10 μM (medium), and 20 μM (high) for further investigations. The results showed that there was a dramatic loss of cell viability and higher cytotoxic effects of SFN on GCs at higher concentrations (>15 μM). The expression of NRF2 increased significantly (p < 0.05) with fold change ranged 3–8 in SFN treated GCs, whereas Kelch Like ECH Associated Protein 1 (KEAP1) expression was either downregulated or similar as control group under the same conditions. Moreover, the relative expression of the genes (PRDX1, CAT, TXN1and SOD1) downstream to NRF2 activation was found to be highly expressed (fold change ranged from 2 to 5, p < 0.05) in SFN treated GCs compared to the untreated control. In addition, ROS accumulation was higher in GCs treated with 20 μM SFN which in turn results in a higher accumulation of lipid droplets. Compared to control, no changes in the mitochondrial activity was observed at 2 and 10 μM SFN concentrations; however, significantly lower mitochondrial activity was found at high concentration (20 μM). The results of this study clearly showed that 10 μM SFN concentration played a crucial role in activating Nrf2 pathway without inducing apoptotic characteristics and this concentration may have beneficial effects in boosting the production of phase II antioxidant enzymes in GCs. However, at high concentration (20 μM), SFN may generate excessive ROS that causes mitochondrial dysfunction and induces cellular stress and eventually leads to apoptosis. These data strongly suggest a concentration dependent antioxidative and apoptotic effects of SFN on GCs.