In recent years, there has been increasing interest in nanoparticles, especially those widely present in our environment. Several studies have been performed to evaluate their potential toxic effect and their possible use for biomedical applications. Among others, cerium dioxide nanoparticles (nanoceria, CeO2 ENPs) have been recently investigated for their use in biomedicine, based on their potential antioxidant function, due to the presence of oxygen vacancies and redox transformations (Ce4+/Ce3+) occurring at the surface. However, little is known about the potential toxicity of nanoceria in the reproductive system and on gametes, and no information is available with regard to its biocompatibility and potential toxicity on male gametes. The aim of the present study was to investigate effects of increasing doses of CeO2 ENPs on ram spermatozoa during 24 h storage at 4°C, based on assessment of main kinematic parameters, membranes and DNA integrity, ROS production, mitochondrial activity, and CeO2 intracellular uptake. The ejaculates of 3 rams of proven fertility were pooled and incubated with increasing doses of nanoceria (0, 22, 44, and 220 µg mL–1) up to 24 h at 4°C. The experiment was conducted in 4 replicates. At 0, 2, and 24 h of incubation, the 4 groups were submitted to the following analyses: i) main kinematic parameters (total motility and progressive motility) through CASA (computer-assisted sperm analysis); ii) acrosome and membrane integrity (propidium iodide + Pisum sativum agglutinin staining, PI+PSA); iii) flow cytometry for sperm chromatin structure assay (SCSA, acridine orange staining), mitochondrial activity (Mitotracker Orange), and ROS production (H2DCFDA). Moreover, an aliquot of semen from each group in each time step was fixed and processed for transmission electron microscopy to assess intracellular uptake of CeO2 nanoparticles by spermatozoa. Increasing concentrations of nanoceria did not affect the main kinematic parameters of ram semen; there were no differences in total and progressive motility among groups at any time point during the 24 h of incubation (P > 0.05). Integrity of the acrosome and cytoplasmic membranes, assessed through PI+PSA staining, was not affected by nanoceria in any group (P > 0.05). Moreover, exposure to nanoparticles did not increase DNA fragmentation (P > 0.05), and there was no difference in the amount of ROS produced and mitochondrial activity within the 24 h of incubation with nanoceria (P > 0.05). Absence of internalization of the nanoparticles by spermatozoa and occasional interaction between the sperm surface and nanoceria were observed by transmission electron microscopy analysis. In the present study, exposure of ram spermatozoa to increasing doses of nanoceria was not cytotoxic; furthermore, high concentrations of these nanoparticles were well tolerated. These data open new perspectives on the biomedical use of nanoceria and provide more information about their impact on male gametes.
Transmission Electron Microscopy of Centrioles, Basal Bodies and Flagella in Motile Male Gametes of Land Plants