Possible involvement of SIRT1 and FOXO3a in oxidative stress response and aging of human granulosa cells

Introduction: It is well known that ovarian function can be compromised by stress conditions induced by physiological events such as reproductive aging and non-physiological events such as chemio- and radiotherapic treatments. SIRT1 is a protomember of the sirtuin family that deacetylates numerous substrates in a nicotinamide adenine dinucleotide-dependent manner. It plays a key role in fundamental cellular processes through its deacetylasic activity on histons, transcriptional factors and cell cycle regulators. The involvement of SIRT1 in the stress response is attributed to its interaction with several molecular targets including members of the FOXO family, forkhead transcription factors which control cell cycle arrest, differentiation, detoxification of reactive oxygen species (ROS), DNA repair and apoptosis. Under stress conditions caused by depletion of nutrients or presence of ROS, FOXOs are dephosphorylated and traslocated to the nucleus, where they can be activated by SIRT1. The activated FOXOs stimulate ROS detoxification through up-regulation of scavenging enzymes such as superoxide dismutase and catalase. The present study was undertaken to determine whether SIRT1 is expressed in human granulosa cells (hGCs) and to investigate the possible involvement of SIRT1 and its target FOXO3a in aging and oxidative stress response. Material and Methods: Human GCs were isolated from follicular fluids of 24 women aged between 28 and 42 years undergoing controlled ovarian stimulation for in vitro fertilization. After cell lysis, protein extracts were subjected to western blotting and analyzed by using antiSIRT1 and antiFOXO3a antibodies. Pooled hGC samples from young (age 28-31) and reproductive old (age 39-42) patient groups were cultured overnight and subjected to immunocytochemistry by using the same antibodies. After incubation with secondary antibody conjugated with Alexa Fluor 488 and Hoechst 33342 labelling, cells were observed under confocal fluorescence microscope. Two groups of young and aged GCs were exposed to 150 µm H2O2 prior to their processing for immunocytochemistry. Results: Western blotting has revealed in hGCs the presence of proteins with molecular weight of about 110 kDa and 70 kDa corresponding to SIRT1 and FOXO3a, respectively. Both proteins were identified in all samples independently of age. Immunofluorescence analysis showed that SIRT1 was localized mainly in the cytosol in both young and aged groups. However, in the young group a significantly higher number of cells had a nuclear localization when compared to aged hGCs. Under stress conditions, in the young group SIRT1 was observed in the nucleus of all cells, whereas in the aged group it maintained a cytoplasmic localization. Our observations revealed that FOXO3a was mainly localized in the nucleus in young and aged group. However in the aged group a significantly higher number of cells showed a cytoplasmic distribution. Under stress conditions FOXO3a was translocated to the cytoplasm in most hGCs of the young group whereas in the aged group it maintained a nuclear localization. Conclusions: Our data showed that stress conditions induced by hydrogen peroxide affect intracellular distribution of both SIRT1 and FOXO3a in hGCs suggesting their involvement in the same pathway. Also, based on the observation that young and aged hGCs exhibited different patterns of distribution before and after H2O2 exposure, it is likely that aging may cause deregulation of both proteins. Moreover, present results represent the first evidence in the literature of SIRT1 expression in hGCs of periovulatory follicles. This finding, along with the observation of a possible interaction between SIRT1 and FOXO3a, can be an important starting point for defining hGC response to stress conditions associated with ovarian ageing and dysfunctions.