118 EXOSOME-MEDIATED OXIDATIVE STRESS RESPONSE IN BOVINE GRANULOSA CELLS

Abstract

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.