The influence of highly dispersed silica nanoparticles on the functional activity of mitochondria and chromatin state in native and devitrified Bos taurus oocytes

Mitochondria are the only cellular compartments which generate and transform energy in the cell. These organelles are among the first to respond to changes in extra- and intracellular conditions (e.g., ionic homeostasis, dehydration level, temperature). Exposition to ultra-low temperatures, due to lipid peroxidation, causes ATP synthetase disorders and destruction of genetic material (E.A. Novocherkina et al., 2016). Highly dispersed silica nanoparticles (nHDS) can be proposed as cytoprotectors. An amorphous form of silicon dioxide, or highly dispersed silica, which exhibits its biological activity through high adsorption capacity, reduces the concentration of ions and biopolymers upon cell dehydration during cryopreservation, thus preventing a sharp change in the osmolar equilibrium between the intra- and extracellular environment (T.T. Turov et al., 2011). This paper deals with the first report of the increase in cryoresistance and maturation rate of donor Bos taurus oocytes due to exposure to nHDS during vitrification and in vitro culture, which ensures higher functional activity of mitochondria and preserves structural properties of chromatin of native and devitrified (DV) oocytes. The study aimed to identify the impact of nHDS on functional activity of mitochondria and chromatin state of cows’ native and devitrified oocytes during in vitro culture. In the experiments, we used oocyte-cumulus complexes (OCCs) of Holsteinized cows. The oocytes with homogeneous cytoplasm surrounded by five or more layers of cumulus cells were subjected to vitrification. OCCs intended for vitrification were successively exposed to three solutions of cryoprotective agents (CPAs) based on T-199 medium with 10 % fetal bovine serum (FBS, HyClone, Great Britain), the CPA-1 containing 0.7 M dimethyl sulfoxide (DMSO) + 0.9 M ethylene glycol (EG) (for 30 s), CPA-2 (1.4 M DMSO + 1.8 M EG (for 30 s), and CPA-3 (2.8 M DMSO + 3.6 M EG + 0.65 M trehalose) (for 20 s). The oocytes in straws were plunged into liquid nitrogen. After thawing, the oocytes were washed with T-199 medium containing 0.25 M, 0.19 M, and 0.125 M trehalose, and finally with T-199 medium. In the test group of oocytes, highly dispersed silica nanoparticles (nHDS, 4-17 nm, mass concentration 0.001 %) synthesized by high-temperature hydrolysis were added to the CPAs, devitrification solutions and washing solutions. In the control group, native and devitrified oocytes were cultured for 24 hours in maturation medium, the T-199 with 106 granulosa cells/ml supplemented with 10 % fetal bovine serum and 50 ng/ml bovine prolactin (38.5 °C, 90 % humidity, and 5 % CO2 atmosphere). In the test group, the maturation medium for native and devitrified oocytes was supplemented with nHDS at a final concentration of 0.001 %. Mitochondrial activity was measured by fluorescence intensity (FI) of MitoTracker Orange CMTMRos (Thermo Fisher Scientific, UK) in µA. In a series of experiments on identification of the nHDS effects on the nuclear maturation of female gametes, oocytes were exposed for 5-10 min to 0.9 % sodium citrate solution and mechanically released from cumulus cells with a preparation needle. Then the cells were placed on a glass slide and fixed with methanol:acetic acid solution (3:1). The dry-air preparations were stained with azure-eosin by Romanovsky-Giemsa method. In DV oocytes influenced by nHDS, the FI of MitoTracker Orange CMTMRos probe increased from 77±6.3 μA to 169±12.8 μA (p function show_eabstract() { $('#eabstract1').hide(); $('#eabstract2').show(); $('#eabstract_expand').hide(); }