MII mouse oocytes
A total number of 56 cryopreserved MII mouse oocyte were used as the subjects of this study. Cryopreserved oocytes were purchased from Embryotech, USA and no experiments were performed on animals. Therefore, no ethical approval was required to work on the cryopreserved mouse oocytes. All experiments performed at the research laboratory of the Clinical Embryology Department at the Medical School, Ninewells and took place from April 2018 to July 2018. At arrival, oocytes were frozen by utilizing a slow freeze method by the supplier (Embryotech, US). According to the supplier, purchased oocytes were harvested from superovulated female B6C3F-1 x B6D2F-1 mice 12 hours post-hCG injection. Before each experiment, oocytes were thawed at the research laboratory of the Clinical Embryology Department at the Medical School, Ninewells according to the supplier instructions. Briefly, straws containing 5-10 oocytes were removed from the cane and held for 1 minute in a 37oC water bath and then removed and wiped dry. The contents of the straw were immediately expelled into a holding dish containing 1 ml of HEPES buffered medium (Origio, Denmark). Finally, after the warming process, oocytes in the holding dish were incubated in a non-gassed incubator at 37oC until use. Only Metaphase II oocytes with normal morphology represented by the presence of zona pellucida, normal cytoplasm, and single polar body were included in this study. Oocytes with abnormal morphology and different maturation stages were excluded from the study subjects.
Oocytes were arbitrarily divided between experimental groups that were studied independently from each other in order to assess Ca2+ and plasmalemmal membrane potential levels:
- Negative control (in the absence of any compound)
- Dimethyl sulfoxide DMSO 0.1% ( used to dissolve glibenclamide and FCCP)
- Glibenclamide (100 μM in 0.1% DMSO, a KATP channel blocker)
- Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) 50 μM in 0.1% DMSO (oxidative phosphorylation inhibitor and chemical hypoxia inducer)
- FCCP 50 μM+ Glibenclamide 100 μM (this group was only included for oocytes stained with Fluo-3 AM )
Laser Confocal Microscopy Imaging
Oocytes were loaded with Fluo-3 AM Calcium indicator (0.5 µg/ml in HEPES medium, at 37˚C for 60 minutes) and Di-8-ANEPPS plasmalemmal membrane potential probe (10 μM in HEPES medium, at 37˚C for 10 minutes). Stains were freshly prepared on the day of the experiments. After staining, oocytes were loaded into the wells of the ibidi slide (ibidi, Germany) containing 180 µl of HEPES buffered media without Human Serum Albumin (to prevent oocyte from floating and moving during the analysis). No more than 5 oocytes were loaded into the slide well. After oocyte loading, the slide was transferred to the confocal laser microscope room by a transport incubator at 37°C. The ibidi slide containing the oocytes was placed on the confocal laser microscope stage inside an environmental chamber. The temperature of the microscope chamber was maintained at 37°C during all the preparation and analysis times. Each oocyte imaged using laser confocal microscopy coupled to an inverted microscope (Leica TCS SP5 II, Milton Keynes, UK) with a ×10 (numerical aperture 1.3) oil-immersion objective lens. The intensity of fluorescence of whole oocytes on the equatorial plane was measured. The microscope was calibrated by the green calibration slide before each experiment. The intensity of fluorescence was described in arbitrary units (AU) covering a range from 0 to 60000 AU. Ca2+ levels, plasmalemmal membrane potential and cell morphology were imaged every 10 min for 2 h using an Argon/UV laser (excitation 480-505 nm/emission 520-610 nm). After the 2 hours, the fluorescent intensity of each oocyte probed with Fluo-3 AM and Di-8-ANEPPS was measured using Leica X Life Science software. Intensity quantification was done by marking the first region of interest which is the oocyte (ROI 01) and duplicating that region elsewhere in the field to represent the measurement background (ROI 02). The intensity of the background (ROI 02) was then subtracted from ROI 01 value to give the true fluorescent intensity measurement in the oocyte. Numerical values in the excel sheet were taken for each oocyte over time by Leica X Life Science software (Leica Microsystems, Germany). For Fluo-3 AM, an increase in the pink fluorescent intensity indicates higher intracellular Ca2+ (Mitochondrial and endoplasmic reticulum Ca2+). For Di-8-ANEPPS, an increase in the green fluorescent intensity indicates plasmalemmal membrane depolarisation, whereas a decrease in fluorescent intensity suggests plasmalemmal membrane hyperpolarisation. This process was repeated for each oocyte and time interval for all the tested groups. The parameters of image acquisition were similar for all examined oocytes. Unless otherwise specified, all reagents and chemicals used in this study were purchased from Sigma-Aldrich.
The normality and assumptions were calculated by using SigmaPlot version 4, from Systat Software, Inc., San Jose California USA to ensure the data were normally distributed. The Shapiro–Wilk statistical test was used for normality testing. Repeated measures two-way Analysis of Variance (RM Two-way ANOVA) was performed as the data had two variables (time and intensity). This was carried out using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA. Additionally, Tukey’s multiple comparisons test was used to detect any statistical significance between the individual groups and the different time points within each group. A P value less than 0.05 was considered statistically significant.