Effects of N-acetylcysteine on growth, viability and reactive oxygen species levels in small antral follicles cultured in vitro

Abstract

Objective: To investigate the effects of different concentrations of N-acetylcysteine on follicular growth and morphology, as well as on viability, levels of reactive oxygen species (ROS) and meiotic progression of oocytes from in vitro cultured bovine early antral follicles.Methods: Isolated early antral follicles (about 500 μm) were cultured in TCM-199+ alone or supplemented with 1.0, 5.0 or 25.0 mM N-acetylcysteine at 38.5 °C with 5% CO2 for 8 days. Follicle diameters were evaluated at day 0, 4 and 8 of culture. At the end of culture, the levels of ROS, chromatin configuration and viability (calcein-AM and ethidium homodimer-1 staining) were investigated in the cumulus-oocyte complexes. Comparisons of follicle diameters between treatments were performed. Data on percentages of morphologically normal follicles, growth rates and chromatin configuration in different treatments were compared.Results: An increase in follicular diameters after culture in all treatments was observed, except for follicles cultured with 25.0 mM N-acetylcysteine. Fluorescence microscopy showed that oocytes cultured in all treatments were stained positively with calcein-AM, and that 5.0 mM N-acetylcysteine reduced fluorescence for ethidium homodimer-1. Intracellular levels of ROS in oocytes from follicles cultured with 1.0 mM N-acetylcysteine showed a significant reduction compared to other treatments. The presence of N-acetylcysteine in culture medium did not influence the rates of oocyte at the germinal vesicle stage.Conclusions: N-acetylcysteine at concentrations of 1.0 and 5.0 mM reduces ROS levels and staining for ethidium homodimer-1 in in vitro cultured follicles, respectively, while 25.0 mM N-acetylcysteine decreases follicular growth and the percentages of continuously growing follicles.