The effect of TiO₂ nanoparticles on antral follicles is dependent on the nanoparticle internalization rate

Titanium dioxide nanoparticles (TiO₂ NPs) are among the most widely produced metallic NPs due to commercial and industrial applications in products including food, cosmetics, paints, and plastics. TiO₂ NPs are released into the environment posing health risks for humans and wildlife. Widespread uses have raised concerns about the potential toxicity of TiO₂ NPs in reproduction. The ovary is an important endocrine organ responsible for sex steroid hormone production and folliculogenesis. NPs can reach the ovary, but limited information is available regarding NP toxicity and its effects on ovarian antral follicles. Thus, we tested the hypothesis that exposure to TiO₂ NP affects sex hormone synthesis, oxidative stress, and antioxidant response in ovarian antral follicles in vitro. In addition, we characterized the NP internalization in the antral follicles over time to determine any association between NP internalization and effects on the antral follicle. Antral follicles were exposed to vehicle control or TiO₂ NPs (5, 25, and 50 μg/ml) for 96 h. The lowest NP concentration (5 μg/ml) showed no internalization and no effects in antral follicles. The 25-μg/ml concentration had the highest internalization rate, leading to increased mRNA ratio of Bax to Bcl2. Interestingly, the highest concentration (50 μg/ml) showed lower internalization compared with the 25 μg/ml, with altered levels of steroidogenic involved genes and increased levels of progesterone and testosterone compared with control. In conclusion, these data suggest that TiO₂ NP is internalized in antral follicles as the first step process in impairing follicle functions.