Effect of exposure to CeO₂ nanoparticles on ram spermatozoa during storage at 4 °C for 96 hours

Over the past decades, nanoparticles of Cerium oxide (CeO₂ NPs) have received considerable scientific interest due to their peculiar chemical, physical and biological properties. However, their everyday use in industry, food science and cosmetics is limited by the concerns about potential effects of their distribution and bioaccumulation in the environment [1]. More recently, the use of CeO₂ NPs in biomedicine has been taken into account considering their capacity to store oxygen and consequent scavenger activity against reactive oxygen species (ROS) comparable to that of antioxidant enzymes in biological systems [2‐4]. In literature, a large number of articles described a reduction in ROS levels in several tissues or cells following exposure to CeO₂ NPs. Recently, scavenger action has been reported in cortical neurons in rats with spinal injuries [5], human keratinocytes [6], mice endothelial cells and fibroblasts [7], human breast and fibrosarcoma cells [8] and cardiac cells [9]. In contrast, many authors reported pro-oxidant effects especially in pulmonary cells [10, 11] and DNA damage in liver cells and leucocytes [12]. These diverging observations suggest that CeO₂ NPs may show the same paradox activity described for other scavenger substances, perhaps depending on the physical and chemical characteristics of the compound, the concentration, the length of exposure and the biological system involved.

Few studies focussed on the effects of CeO₂ NPs in the reproductive system and the findings are often in contrast. In mice, exposure of oocytes to increasing doses of CeO₂ NPs led to oxidative stress and consequent DNA damage [13]. In the same species, a decrease in fertilization rates and accumulation in granulosa cells and sperm plasma membranes has been described [14]. However, in the ovine, gametes well tolerated co-incubation with CeO₂ NPs. In particular, our research group reported that granulosa cells but not oocytes internalise this compound by endocytosis. Moreover, low concentrations of NPs enhanced in vitro fertilization of oocytes with low developmental competence possibly throughtheir scavenging action and downregulation of genes activated by oxidative stress [15].

Ram spermatozoa exposed to increasing concentrations of CeO₂NPs for 24 h, showed no NPs uptake, occasionally sporadic contacts with plasma membranes and no adverse effects on DNA integrity and motility parameters. Furthermore, the redox balance of the cells was not perturbed by exposure to NPs since both ROS levels and mitochondrial activity remained stable [16]. We hypothesised that, submitting ram spermatozoa to a prolonged stressing condition such as extended storage at 4 °C and exposing them to CeO₂ NPs would have triggered the scavenging action of this compound. Thus, the aim of the study was to investigate the effects of the exposure to increasing doses of CeO₂ NPs on the kinematic parameters, integrity of membranes, DNA fragmentation and oxidative status of ram semen stored at 4 °C for 96 h.

In this study, we reported the effects of exposure to CeO₂ NPs of ram spermatozoa stored for 96 h at 4 °C. The choice of these experimental conditions was based on the hypothesis that CeO₂ NPs, with their catalytic action, might prevent the deleterious effects of storage at low temperatures on ram sperm cells. The decrease in quality of refrigerated semen restricts its application during artificial insemination programs to a short time span, limiting the diffusion of superior genotypes to small geographic areas and to restricted numbers of selected animals. For this reason, the perspective of storing semen for longer times would represent a great benefit in the management of genetic selection and reproduction in sheep breeding systems and a valuable alternative to cryopreserved semen.

Over the past decades many research groups focussed on improving the quality of refrigerated ram semen. The most promising approach is represented by the supplementation of storage extenders with anti-oxidant substances that contrast the deleterious effects of ROS accumulation and oxidative stress on sperm cells. Among others, CeO₂ NPs are gaining increasing interest for their ability to change their oxidation status [19] conferring them promising scavenger properties.

Our research group [16] previously reported that ram spermatozoa stored at 4 °C for 24 h well tolerated concentrations of CeO₂ NPs above those commonly dispersed in the environment (water 0.024 mg/L [20]; soil 1.12 mg/kg [21]) and the results reported in the present study are in agreement with these observation. The potential toxic effects of these compounds have to be carefully assessed and the consequences on the reproductive system are still under investigation. In mice, low concentrations of CeO₂ NPs significantly affected in vitro fertilization and had genotoxic effects on both male and female gametes [13, 14]. In the present study, we can state that none of the sperm parameters analysed was negatively affected by extended exposure of sperm cells to CeO₂ NPs.

Moreover, we described a consistent and significant increase in kinematic parameters of spermatozoa incubated with high concentrations of NPs that has not been reported before. This beneficial effect was mostly evident in spermatozoa exposed to 220 μg/mL NPs from 48 h of incubation onward. In fact, in the first 24 h of the trial, CeO₂ NPs did not exert any effect on any of the analysed parameters. It has been reported that the overall quality of ram semen stored at 4 °C dramatically drops after 3 days [22‐24] and we can speculate that the activity of NPs might be stronger in cells with compromised morphologic and structural characteristics under stressing conditions such as storage at low temperatures.

In addition to the enhancement of TM and PM, incubation with NPs promoted the increase in parameters related to cells velocity (VAP, VCL and VSL) of around 10 μm/s. It has been reported that these parameters are highly correlated to fertility in several species such as ram [25, 26], bull [27] and boar [28]. Incubation with NPs also changed the rates of rapid spermatozoa in the exposed groups compared to the control with more than 50% of cells having a VAP > 75 μm/s at 72 and 96 h of incubation. These results are in agreement with what previously reported in rats fed with a diet containing citrate-coated CeO₂ NPs (1 mg/kg). After 10 days of diet, the authors reported a significant increase in motility and viability of epididymal spermatozoa in treated animals compared to control group and suggested NPs as a helpful tool in contrasting age-related infertility [29]. A recent study carried out on humans reported an improvement in motility parameters in frozen- thawed semen following supplementation of cryopreservation medium with ZnO NPs [30]. These promising results may pave the way for a use of NPs as preservers of semen quality during storage at low temperatures although further investigations are strictly needed. Preaubert et al. reported no effect of CeO₂ NPs on progressive motility rates in mice spermatozoa [14] and detrimental effects of NPs on sperm motility have been described in bull [31] and human [32], suggesting species-specific effects of these compounds.

Data on motility correlated well with the results obtained by the analysis of the integrity of plasma and acrosomal membranes suggesting a high biocompatibility of CeO₂ NPs. As for kinematic parameters, NPs did not influence the status of the membranes in the first 48 h of exposure, but from 72 h onward, the effects of co-incubation were visible on spermatozoa exposed to NPs, that preserved more efficiently the morphologic structure of plasma membranes compared to unexposed cells. Acrosomes were not affected by exposure to NPs and this suggests a differential sensitivity of sperm membranes to stressing conditions. The absence of membrane alterations during incubation with NPs has been previously observed in the same species [16]. Moreover, no interaction with membranes or up-take but only occasional contacts between NPs and the post-acrosomal region of spermatozoa was described [16], suggesting that the positive effects triggered by NPs on sperm parameters are not mediated by intra-cellular mechanisms.

In this experiment, we exposed spermatozoa to a prolonged stressing factor (storage at 4 °C for 96 h) and investigated the role of CeO₂ NPs in preventing oxidative sperm damage. However, the levels of ROS remained fairly stable until 72 h and then rose at 96 h in exposed and unexposed cells.

During storage procedures at low temperatures, ram spermatozoa are extremely sensitive to oxidative stress due to the high ratio unsaturated/saturated fatty acids in phospholipids of plasma membranes [33]. Disruption of intra-cellular balance of free radicals accumulation leads to impairment of quality parameters such as viability, motility, membranes integrity and consequent fertilizing ability, as reviewed by Bansal and Bilaspuri [34]. On the other side, ROS play a critical role in several essential physiological processes such as sperm binding, capacitation and hyper-activation, as recently reviewed by O’Flaherty et al. [35].

Although we reported a positive effect of CeO₂ NPs in preserving quality traits in semen from 48 h onward, we could not correlate this effect with changes in the levels of ROS. This controversial result suggests that NPs, in the specific experimental conditions of the present trial, acted through pathways that are independent from ROS accumulation in sperm cells.

We could also speculate that CeO₂ NPs might exert their catalytic action in the extra-cellular compound (storage extender) perhaps through their SOD or catalase mimetic behaviour, rather than inside sperm cells. This hypothesis is supported by the previously reported lack of uptake and internalisation of CeO₂ NPs by sperm cells [16]. These external actions possibly depend on chemical and physical characteristics of NPs and interaction with the compounds involved in storing spermatozoa, such as extender and seminal plasma. In physiological conditions, SOD and catalase antioxidant activities have been detected in seminal plasma of several species like bull [36], boar [37] and equine [38], where they are involved in maintaining integrity of membranes and sperm function. We can also suggest that supplementation of storage extender with CeO₂ NPs might have supported the activity of the enzymatic systems formerly present in seminal plasma of rams.

Perrin et al. reported that exposure of human spermatozoa to CeO₂ NPs has genotoxic effects that were limited by the use of an antioxidant (L-Ergothioneine) suggesting a pro-oxidant activity of the NPs [39]. In our experiment, we did not report any genotoxic effect of CeO₂ NPs at any time of the experimental trial. The levels of DNA fragmentation remained stable for up to 96 h and no differences were found with unexposed spermatozoa. This indicates that the biocompatibility of these compounds may depend by several factors; among others, a species sensitivity should not be excluded. In the mouse, CeO₂ NPs induce DNA damage in both oocytes and spermatozoa affecting in vitro fertilization [14] whereas in the ovine species, supplementation of maturation media with CeO₂ NPs (44μg/mL) did not impair but enhanced fertilization and blastocyst rate and no adverse effects were observed in chromatin configuration of oocytes exposed to NPs [15]. On the other side chromatin damage has been reported in mice [40] and bull spermatozoa [41] exposed to silver NPs.

To our knowledge, this is the first report describing beneficial effects of CeO₂ NPs on morphologic and kinematic parameters of ram semen, such as motility and plasma membrane integrity after 96 h of exposure. We also reported no genotoxic effects of these NPs. However, these beneficial effects could not be explained by an intra-cellular antioxidant activity exerted by these compounds, since ROS levels in exposed cells were similar to those of unexposed ones. Eventually, further investigations are needed to support these preliminary results and to pave the way to future applications of these compounds in reproductive biology.