Evaluation of developmental competence of Sus scrofa domesticus (L.) oocyte-cumulus complexes after intra- and extraovarian vitrification

The aim of the present study was to identify the influence of extra- (EOV) and intraovarian vitrification (IOV) on mitochondrial activity (MA) and chromatin state in porcine oocytes during maturation in vitro. During EOV porcine oocytes were exposed in cryoprotective solutions (CPS): CPS-1 – 0.7 M dimethyl sulfoxide (DMSO)+0.9 M ethylene glycol (EG); CPS-2 – 1.4 M DMSO+1.8 M EG; CPS-3 – 2.8 M DMSO+3.6 M EG+0.65 M trehalose. At IOV the ovarian fragments were exposed in CPS-1 – 7.5 % EG+7.5 % DMSO, then in CPS-2 – 15 % EG, 15 % DMSO and 0.5 M sucrose. Straws with oocytes and ovarian fragments were plunged into LN2 and stored. For devitrification, the EOV oocytes were washed in solutions of 0.25, 0.19 and 0.125 M of trehalose, the IOV – in 0.5 and 0.25 М trehalose. Oocytes were cultured in NCSU-23 medium with 10 % fluid of follicles, follicular walls, hormones. 0.001 % of highly dispersed silica nanoparticles (ICP named after A.A. Chuyko of the NAS of Ukraine) were added to all media. The methods of fertilization and embryo culture are presented in the guidelines developed by us. MA and chromatin state were measured by MitoTracker Orange CMTMRos and the cytogenetic method. Significant differences in the level of oocytes with high-expanded cumulus between control and experimental vitrified groups (81 % versus 59 % and 52 %, respectively, p ≤ 0.001) were observed. The percentage of pyknotic cells in native oocytes was 19 %, EOV or IOV oocytes were 39 % and 49 %, respectively. After culture, the level of matured native oocytes was 86 %, 48 % EOV and 33 % IOV cells finished the maturation (p ≤ 0.001). Differences were also observed in the level of MA between groups treated by EOV and IOV (89.4±7.5 µA and 149.2±11.3 µA, respectively, p ≤ 0.05). For the first time, pre-implantation embryos were obtained from oocytes treated by IOV.

Development of a live cell imaging method for mitochondria in Trebouxia photobionts

The Trebouxia photobiont freshly isolated from Xanthoria parietina (L.) Th. Fr. was used to develop a live cell chondriome (mitochondrial DNA) labelling method. In the initial phase six candidate dyes were tested and compared for mitochondrial labelling utility as assessed by the signal to noise ratio (SNR) of the mitochondrial signal to the adjacent cellular background in standardized confocal images of 30 labelled cells. DIOC7, JC-1 and MitoTracker orange (MTO) dyes showed some labelling ability. MTO had significantly higher utility than the other dyes. In a second phase, MTO concentration was optimized. The final labelling protocol was a 30 minute incubation with 1 μM of MTO. The resultant labelling was suitable for both widefield and confocal microscopy. Both 2D thresholding and 3D volume construction are demonstrated using the resultant data. The protocol can therefore be utilized for both qualitative research and for quantitative measurement of the chondriome in Trebouxia photobionts. This will facilitate a wide range of mitochondrial investigations in lichenology.

Real-Time Measurement of Intracellular Reactive Oxygen Species Using Mito Tracker Orange (CMH2TMRos)

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH2TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120–150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH2TMRos. Incubation with glucose oxidase, exogenous H2O2, KO2 and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH2TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH2TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H2O2.