Effects of N-acetylcysteine on Growth, Viability, and Ultrastructure of In Vitro Cultured Bovine Secondary Follicles

Abstract

This study aimed to investigate the effects of different concentrations of N-acetylcysteine (NAC) on the growth, antrum formation, viability, and ultrastructure of bovine secondary follicles cultured in vitro for 18 days. To this end, the follicles were cultured in TCM-199+ medium alone or supplemented with 1.0, 5.0, or 25.0 mM NAC. Follicular growth, antrum formation, viability (calcein-AM and ethidium homodimer-1) and ultrastructure were evaluated at the end of culture period. The results showed that 1.0 mM NAC increased the percentage of growing follicles and the fluorescence intensity for calcein-AM when compared to other treatments (p < 0.05). On the other hand, follicles cultured with 25.0 mM NAC had higher fluorescence intensity for ethidium homodimer-1, which is a sign of degeneration. Ultrastructural analysis showed that oocytes from follicles cultured in control medium alone or with 1 mM NAC had intact zonae pellucidae in close association with oolemmae, but the ooplasm showed mitochondria with a reduced number of cristae. On the other hand, oocytes from follicles cultured with 5 or 25 mM NAC had extremely vacuolated cytoplasm and no recognizable organelles. In conclusion, 1 mM NAC increases cytoplasmic calcein staining and the growth rate in bovine secondary follicles cultured in vitro, but the presence of 5 or 25 mM NAC causes damage in cellular membranes and organelles, as well as reducing the percentages of growing follicles.

Results of Ultrasound Studies of the Growth Dynamics of Dominant, Subdominant Follicles and Determination of Estradiol Concentration in the Preovulatory Period in Cows

“…According to the results of the study, the volume of mitochondria occupied by lipid droplets was higher (P<0.03) in oocytes from regressing follicles (193.0±10.4/1000microns 3 and 3.5±0.7%) than in the growth stage and in the preovulatory stage (118.7±14.4/ 1000microns 3 and 1.1±0.3%; 150.5±28.7/1000microns 3 and 1.6±0.2%, respectively). Thus, it was found that lipid droplets undergo spatial reorganization, i.e., from peripheral to uniform distribution during the growth phase and the mitochondrial lipid contact area increases as the follicle matures (Dadarwal et al 2015;Fayezi et al 2018;Mylostyvyi et al 2021;Azevedo et al 2022;Nascimento et al 2022).…”

Impact of Thermo-Responsive N-Acetylcysteine Hydrogel on Dermal Wound Healing and Oral Ulcer Regeneration

“…In said study, the combination of NAC with curcumin, despite its potential as an antioxidant and antifibrotic agent, showed no significant improvement in cell viability, further complicating its application. This was even exemplified in studies exploring the NAC's capacity to protect oocytes [27] and endothelial cells [28]. In the former study, NAC has been shown to be beneficial when used at 1.0 mM concentration but can significantly affect cellular ultrastructure and reduce the viability of bovine secondary follicles when used at concentrations greater than 5.0 mM [27].…”

“…This was even exemplified in studies exploring the NAC's capacity to protect oocytes [27] and endothelial cells [28]. In the former study, NAC has been shown to be beneficial when used at 1.0 mM concentration but can significantly affect cellular ultrastructure and reduce the viability of bovine secondary follicles when used at concentrations greater than 5.0 mM [27]. In the latter study about endothelial cells, although low concentrations of NAC have been shown to offer protection against oxidative stress induced by proinflammatory cytokines, high concentrations trigger a biphasic response, leading to increased ROS production, protein carbonylation, and glutathionylation, highlighting the complex interplay between antioxidants and cellular responses [28].…”

“…All formulations were tested at 50:50% volume solutions of NAC and MC. Taking the results of the in vitro test conducted with the commercial NAC solution, we opted to utilize lower concentrations of NAC for the resulting hydrogel to avoid the biphasic effect of NAC at higher concentrations [24,[26][27][28]. Results of the gelation test indicated that all formulations were capable of forming a stable gel with increased temperature.…”

Melatonin improves the viability and ultrastructure of bovine oocyte‐granulosa complexes of in vitro cultured early antral follicles

“…In the last decades, in vitro culture of preantral follicles has raised great scientific interest as an alternative to have competent oocytes for in vitro embryo production (Chaves et al., 2011). However, in bovine species, the most promising results achieved are limited to follicular growth up to antrum formation (Nascimento et al., 2022; Paulino et al., 2022). In primates, however, in vitro culture of early antral follicles resulted in yield of competent oocytes (rhesus monkeys: Peluffo et al., 2010, baboons: Xu et al., 2011).…”