Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 μM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative stress released exosomes enriched with mRNA of Nrf2 and candidate antioxidants. Subsequent co-incubation of StressExo with cultured granulosa cells could alter the relative abundance of cellular oxidative stress response molecules including Nrf2 and antioxidants CAT, PRDX1 and TXN1. The present study provide evidences that granulosa cells exposed to oxidative stress conditions react to stress by activating cascades of cellular antioxidant molecules which can also be released into extracellular environment through exosomes.

Exosomes are nano-sized (30–100 nm) extracellular membrane vesicles released through exocytosis process in most cells and biological fluids. They contain a cargo of nucleic acids, proteins, lipids and play a vital role in cell-cell communications. Various cell types have been shown to release exosomes into extracellular space as a response to various environmental stress conditions. However, little is known about the response of granulosa cells to oxidative stress, with regard to release of exosomes that may carry mRNA and protein molecules related to cellular oxidative stress response. Here we aimed to investigate the potential release of stress elements by granulosa cells to culture media through exosomes under oxidative stress conditions. For that, bovine granulosa cells from small follicles were aspirated and cultured in DMEM/F-12 media supplemented with exosome free fetal bovine serum (Exo-FBS) and treated with 5 µM H2O2 for 40 min. Granulosa cells were collected 24 h post-treatment to quantify the expression of antioxidants (Nrf2, Keap1, SOD1, CAT1, PRDX1, HOMOX1, TXN1, and NQO1), cell proliferation (PCNA and CNND2), cell differentiation (CYP11A1 and STAR), apoptosis (Casp3), and antiapoptosis (BCL2L1) genes. Reactive oxygen species accumulation, mitochondrial distribution, cell viability, and cell cycle assays were performed in cultured granulosa cells, and the culture medium was used to isolate exosomes using ultracentrifugation procedure. The identity of exosomes was confirmed by immunoblotting of Alix and CD63 proteins, and the expression level of antioxidant was analysed in mRNA isolated from exosomes. Data from 3 independent biological replicates were statistically analysed using the 2-tailed t-test. Results showed that H2O2 treatment increased mRNA and protein level of antioxidants (Nrf2, PRDX1, and TXN1), as well as cell differentiation and apoptosis-related genes compared to untreated controls. However, granulosa cells treated with H2O2 showed lower expression of cell proliferation marker genes (PCNA and CNND2). Cells treated with H2O2 showed increases in reactive oxygen species level, inadequate mitochondrial distribution, and lower cell viability. Cell cycle assay revealed a reduction in G0/G1 proportion and increase in G2 phase in cells treated with H2O2. Higher levels of antioxidant (Nrf2, CAT1, and TXN1) transcripts were detected in exosomes isolated from media with cells under oxidative stress conditions compared to the controls. Labelling and co-transfection of exosomes from stressed cell culture medium with untreated treated recipient granulosa cells resulted in increased abundance of cellular mRNA and protein of Nrf2 and CAT1 in those cells. In conclusion, granulosa cells exposed to oxidative stress could release exosomes that carry molecules related to oxidative stress response, which can be up taken by recipient cells.