Elsevier

Theriogenology

Volume 85, Issue 7, 15 April 2016, Pages 1274-1281.e3
Theriogenology

Research article
Cerium dioxide nanoparticles did not alter the functional and morphologic characteristics of ram sperm during short-term exposure

https://doi.org/10.1016/j.theriogenology.2015.12.011Get rights and content

Abstract

The aim of the study was to investigate the interaction and the short-term effects of increasing doses of cerium dioxide nanoparticles (CeO2 NPs) on ram spermatozoa, stored at 4 °C for up to 24 hours, on the main functional and kinematic parameters. Spermatozoa were incubated with 0, 22, 44, and 220 μg/mL of CeO2 NPs at 4 °C and submitted at 0, 2, and 24 hours to the following analyses: (1) intracellular uptake of CeO2 NPs by the spermatozoa; (2) kinematic parameters; (3) acrosome and membrane integrity; (4) integrity of DNA; (5) mitochondrial activity; (6) ROS production. The results indicated that the exposure of spermatozoa to increasing doses of nanoceria was well tolerated. No intracellular uptake of NPs by the cells was observed and both kinematic parameters and status of the membranes were not affected by the incubation with NPs (P > 0.05). Moreover, no influence on the redox status of spermatozoa and on the levels of fragmentation of DNA was reported among groups at any time (P > 0.05). The data collected provide new information about the impact of CeO2 NPs on the male gamete in large animal model and could open future perspectives about their biomedical use in the assisted reproductive techniques.

Introduction

There is an increasing interest toward nanoparticles and their potential applications in everyday life. Engineered NPs are defined by the SCENIHR (Scientific Committee of Emerging and Newly Identified Health Risk) as particles which size ranges from 1 to 100 nm in diameter, conferring them novel chemical, physical, and biological characteristics. Because of their peculiar physicochemical properties, NPs are also nowadays widely studied for their use in biomedicine, e.g., as drug delivery systems, drug targeting, diagnostic imaging, and for treatments of a wide range of pathologies [1].

However, a better knowledge of their effects at cellular and tissue levels is necessary, and little information is available about their activity in the reproductive system. It has been demonstrated that NPs can accumulate in the ovaries [2] and can cross the hemato-testicular barrier [3], [4], [5]. However, their potential effects have not been clearly demonstrated, and they may depend to the chemical composition, cell type, and species sensitivity. Recently, the effects of gold and silver NPs on porcine oocytes and spermatozoa have been evaluated. Gold NPs did not have any effect on oocytes-cumulus complex and spermatozoa, whereas silver NPs were detrimental for oocytes-cumulus complex but not for spermatozoa [6]. Similarly, gold and silver NPs did not affect vitality and motility parameters of human spermatozoa [7]. On the contrary, bovine spermatozoa incubated with gold NPs displayed alterations in the chromatin decondensation ability [8], and exposure to silver led to cytotoxicity and genotoxicity of testicular cells in the mouse [9].

Among others, cerium dioxide, an inorganic compound of cerium, a rare earth element of lanthanide series, and its NPs (cerium dioxide nanoparticles [CeO2 NPs]) are recently exerting an interest for their industrial applications such as catalysis, solid oxide fuel additives, sensor technology, and UV filters. This great interest is also related to the high surface area, which confers to CeO2 NPs the ability to change the oxidation state [10]. This property is comparable to that of biological antioxidants such as superoxide dismutase [11], [12] and catalase [13]. This unique scavenger property of CeO2 NPs [10] has been recently investigated in several biological systems and cell types, representing promising applications in biomedicine [14], [15], [16], [17]. On the other side, negative effects of CeO2 NPs in several cell types of have been reported [18], [19], [20], [21] indicating that this compound, as many other free radical scavenging substances, can have a paradox activity.

Little is known about the potential effects of CeO2 NPs in the reproductive system and gametes. In female germ cells of mice, it has been reported that CeO2 NPs are able to penetrate into follicular cells and to be trapped in zona pellucida inducing dose-dependent oxidative stress and consequent DNA damage [22]. Moreover, a recent study that investigated the effects of CeO2 NPs on IVF in mice, reported genotoxicity in female and male gametes and a deleterious effect on fertilization rates [23]. Conversely, in rats, it has been shown that the supplementation of CeO2 NPs in the diet leads to an improvement in progressive motility (PM) and viability of spermatozoa through an antioxidant activity [24].

To our knowledge, no reports are available nowadays on the interactions and biocompatibility of CeO2 on spermatozoa in large animal model. Therefore, the aim of the present study was to investigate the interaction and the short-term effects of increasing doses of CeO2 NPs on ram spermatozoa, stored at 4 °C for up to 24hour, on the kinematic parameters, membrane status, DNA fragmentation, mitochondrial activity, and reactive oxygen species (ROS) production.

Section snippets

Synthesis and characterization of cerium dioxide nanoparticles (CeO2 NPs)

Cerium (III) nitrate hexahydrate (Ce[NO3]3 6H2O, ABCR 99.9%), urea (CH4N2O, Aldrich 99%), 2-propanol (99.7%, Carlo Erba), 1-M hydrochloric acid (HCl, Aldrich), 5-M aqueous ammonia (NH4OH, Aldrich) were used as received without further purification. Urea was used as coordinating agent, NH4OH and Ce(NO3)3 as inorganic precursors. 7×103 mg of Ce(NO3)3 6H2O were dissolved in 20 mL of 2-propanol, then 0.5 mL of hydrochloric acid were added to it and left under stirring until a homogeneous solution

Results

The results obtained by XRD pattern showed that nanoceria had a cubic fluorite structure of cerianite with a crystal size of 9 ± 1 nm, which is in accordance with the nanoparticle diameter measured by TEM characterization (8.5 ± 1.3 nm; [27]). The nanoparticles aggregation and hydrodynamic radius have been evaluated as a function of concentration (22, 44, and 220 μg/mL) and incubation time (0, 2, and 24 hours). Concentrations of CeO2 of 22 and 44 μg/mL in medium allowed obtaining a suspension

Discussion

To our knowledge, the present study is the first reporting the effects of increasing concentration of cerium dioxide nanoparticles (CeO2 NPs) on mammalian spermatozoa during 24 hours of exposure at 4 °C. The results of this study showed that after incubation of ram semen with different concentrations of CeO2 NPs, no intracellular localization of nanoparticles into the sperm cells was observed. In addition, the exposure to nanoceria did not affect the motility parameters, and no cytotoxicity and

Acknowledgments

The present work has been supported by Fondazione Banco di Sardegna and Centro di Competenza di Biodiversità Animale RAS. The authors would like to thank Dr. G.P. Epifani of Servizio Zootecnico, AGRIS, Agenzia Regionale per la Ricerca in Agricoltura, Bonassai, Italy, for the assistance in providing the animals for the experiment; Mr. S. Marceddu, CNR Istituto Scienze Produzioni Alimentari (ISPA), U.O., Italy, for his assistance in TEM analysis; Dr. S. Garroni, Dipartimento di Chimica,

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