Ti2eu

Apoptosis was analyzed using a TUNEL Assay Kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer’s instructions. Briefly, blastocysts on day 7 were washed four times with 0.1% polyvinyl alcohol in phosphate-buffered saline (PBS-PVA) and fixed with 3.7% paraformaldehyde in PBS-PVA for 30 min. Then, the embryos were permeabilized by an additional incubation in 0.1% Triton X-100 at room temperature (RT) for 40 min. Next, the embryos were incubated with 10% terminal deoxynucleotidyl transferase enzyme and fluorescein-conjugated dUTP (Roche Diagnostics, Indianapolis, IN, USA) at RT for 1 h in the dark. Subsequently, the embryos were incubated with 10 mg/mL of Hoechst 33342 at RT for 10 min to label the nuclei. Finally, the embryos were washed four times with PBS-PVA and mounted on a glass slide. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) and ImageJ software (NIH, Bethesda, MD, USA) were used to analyze the number of fluorescent cells. The apoptosis rate was calculated as the ratio of TUNEL-positive cells to the total number of cells.

The day-7 embryos were collected and fixed with 3.7% paraformaldehyde in PBS-PVA for 30 min, and then permeabilized with PBS-PVA containing 0.1% Triton X-100 at RT for 30 min. Afterward, the embryos were incubated in PBS-PVA containing 3% BSA at RT for 1 h and then incubated with primary antibody LC3B antibody (#ab48394, diluted 1:200; Abcam, Cambridge, MA, USA) and rabbit polyclonal anti-NRF2 antibody (#ab31163, diluted 1:200; Abcam, Cambridge, MA, USA) at 4 °C for 14 h. After washing thrice with PBS-PVA, the cells were incubated with goat anti-rabbit IgG (#ab150077, diluted 1:500; Abcam, Cambridge, MA, USA) for 1 h, followed by incubation with 10 mg/mL of Hoechst 33342 at RT for 10 min to label the cell nuclei. Finally, the embryos were washed four times with PBS-PVA and mounted on glass slides. The number of microtubule-associated protein 1 light chain 3 (MAP1LC3) spots and the fluorescence intensity of Nrf2 were detected using an inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan), and the fluorescence intensity was analyzed with ImageJ software (NIH).

MII oocytes were then incubated in DPBS containing 200 nM MitoTracker Red CMXRos (Beyotime, Shanghai, China) for 1 h to measure mitochondrial abundance. Then, oocytes were co-incubated in DPBS with 15 μg/mL JC-1 (Beyontime, Shanghai, China) for 16 h to measure MMP. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) was used to detect fluorescence signals, and the ratio of red to green fluorescence was examined by ImageJ software (version 8.0.2, NIH, Bethesda, MD, USA).

Cell proliferation was measured using the BeyoClick EdU-555 Cell Proliferation Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. In short, on day 6, the embryos were transferred to fresh IVC medium containing 10% EdU with or without WDL and then incubated at 38.5 °C in an atmosphere of 5% CO₂ and 100% RH for 8 h. Next, the blastocysts were fixed with 3.7% paraformaldehyde in PBS-PVA for 30 min, and then permeabilized in 0.1% Triton X-100 at RT for 30 min. After washing four times with PBS-PVA, the embryos were incubated with 5% BeyoClick Additive Solution at 38.5 °C in an atmosphere of 5% CO₂ and 100% RH for 14 h. Then, the embryos were incubated with 10 mg/mL of Hoechst 33342 at RT for 10 min to label the nuclei. Finally, the embryos were washed four times with PBS-PVA and mounted on glass slides. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) and ImageJ software (NIH) were used to count the number of EdU-positive cells and the total cells. The proliferation rate was calculated as the ratio of EdU-positive cells to the total number of cells.

The oocytes in the 4-cell stage are also used to detect the mitochondrial membrane potential (MMP); the oocytes were incubated in PBS-PVA containing 10 µg/mL 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzi-midazolylcarbocyanineiodide iodide (JC-1, Beyotime, Shanghai, China) in the dark at 38.5 °C in an atmosphere of 5% CO₂ for 17 h. After the oocytes were washed four times with PBS-PVA, the stained oocytes were put into 3 µL droplets of PBS-PVA. We used an inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) to detect the red/green fluorescence signals and analyze the fluorescence intensities with ImageJ software. The average mitochondrial membrane potential (MMP) of the oocytes was calculated as the ratios of red fluorescence intensity (J-aggregates) to green fluorescence intensity (J-monomers). We tested MMP by collecting 10 to 15 oocytes at a time in the 4-cell developmental phase, and each group was repeated more than three times.

The manufacturer’s instructions were followed to analyze cell proliferation using the BeyoClick EdU-555 Cell Proliferation Assay Kit (Beyotime, Shanghai, China). In short, on day 6 of the oocyte culture, 10% EdU was added to the IVC culture medium with or without CHE and then incubated in the dark at 38.5 °C in an atmosphere of 5% CO₂ for 10 h. After incubation, the blastocysts developed to day 7 were washed four times with PBS-PVA, fixed in PBS-PVA containing 3.7% paraformaldehyde for 30 min, and then containing 0.1% Triton X-100 permeabilized for 30 min, all at room temperature. After permeabilization, the blastocysts were washed four times, mixed with 5% BeyoClick Additive Solution, and incubated in the dark at 38.5 °C in an atmosphere of 5% CO₂ for 15 h. Finally, the blastocysts were incubated with 10 µg/mL Hoechst 33,342 in the dark at 37.5 °C for seven minutes to mark the nuclei. An inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) and ImageJ version 8.0.2 software (NIH, Bethesda, MD, USA) were used to count the number of EdU-positive cells and total cells. We compared the level of cell proliferation by calculating the percentage of EdU-positive cells in the total number of blastocysts. The number of blastocysts on day 7 for the EDU staining assay was 15 to 20, and the assay was repeated more than three times.

After 42–44 h, ROS and GSH levels of MII oocytes were detected through H2DCFDA (Beyotime, Shanghai, China) and CMF2HC (Beyotime, Shanghai, China). Each group was stained by H2DCFDA and CMF2HC for 10 μM and 30 min at room temperature, then 4 μL PBS droplets were placed in a 35 mm small dish (Thermo Fisher Scientific, Wilmington, DE, USA), and each group of oocytes was transferred to different dishes in turn. Stained samples were imaged with an inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) using ImageJ software (version 8.0.2, NIH, Bethesda, MD, USA) to examine fluorescence intensity. For each experimental group, 30 to 60 oocytes were selected for analysis.