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.

Summary Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed.