PSI-21 Zinc oxide nanoparticles and their impacts on the normality, activation, development, viability, and oxidative stress of in vitro cultured preantral ovarian follicles.

Abstract The objective of this study was to evaluate the normality, activation, development, viability, and oxidative stress of in vitro cultured preantral ovarian follicles supplemented with zinc oxide nanoparticles (ZnO-np). Zinc is essential for ovarian development and hormonal regulation, influencing oocyte maturation and follicular dynamics. The ZnO-np have the potential to elucidate possible effects on ovarian physiology. They can cross biological barriers and interact directly, potentially influencing ovarian cells in processes such as morphology, activation, development, viability, and cellular oxidative stress. However, the toxic or beneficial effects of these nanoparticles have not yet been fully elucidated. It is also worth noting that the ovine ovary is a translational model for human reproductive research, aiding in the understanding of biological mechanisms. Twelve ovine ovaries were collected for the study from sheep aged between one and 2 years and various stages of the estrous cycle. In the laboratory, superficial fragments of the ovarian cortex were dissected and cut into slices of 9 mm3. Two ovarian fragments were designated for the non-cultured control (D0), while the remaining fragments were subjected to in vitro culture under different conditions: Minimum Essential Medium Eagle supplemented (α-MEM; cultured control); α-MEM supplemented with ZnO-np, and micrometric ZnO (ZnO-m), both at a concentration of 50 μg/mL. The fragments were cultured in 24-well plates and incubated for 1 or 7 days (D1 and D7) at 37°C in a 5% CO2 atmosphere, and complete medium changes on days 2, 4, and 6 (D2, D4, D7). After culture, the fragments were subjected to histological analysis for morphological evaluation and examined under optical microscopy at 400x magnification. The organization of granulose cells, preservation of the basal membrane, oocyte integrity, and follicular classification were evaluated. Cell viability was analyzed through mechanical isolation, while the detection of reactive oxygen species (ROS) was performed using dichlorodihydrofluorescein diacetate (DCFH- DA) under confocal microscopy. Finally, the antioxidant capacity of the culture medium was determined using the ORAC (Oxygen Radical Absorbance Capacity) assay through spectrophotometry. Data on normality, activation, development, ROS, and ORAC of preantral ovarian follicles were analyzed by ANOVA (P <0.05), with means compared using Dunnett and Tukey’s tests (SAS 9.4). Follicle viability was assessed using chi-square analysis. The results demonstrated that fragments cultured with 50 μg/mL of ZnO-np exhibited a higher rate of activation and follicular development (P <0.05), highlighting the positive effect of nanoparticles on follicular development dynamics. Furthermore, fragments treated with ZnO-np exhibited a lower relative fluorescence intensity (RFI) compared to the control group (P <0.05), suggesting that the nanoparticles contributed to mitigating oxidative stress induced by in vitro culture. Additionally, the culture medium exhibited higher antioxidant capacity when supplemented with ZnO-np at seven days of culture. It is concluded that ZnO-np at a concentration of 50 μg/mL do not cause direct cellular damage. On the contrary, they exhibit a beneficial effect on morphology preservation, follicular activation, and development, as well as exerting an antioxidant action, promoting the in vitro culture of preantral ovarian follicles.