Fig 1.
Effects of Brusatol on mouse oocyte maturation.
A) Quantitative analysis of GVBD and Pb1 extrusion in different treatment groups (control, 20 nM, 50 nM, 100 nM, 200 nM and 1000 nM). B) Polar body extrusion failure after Brusatol treated. Images were acquired with a camera on a stereomicroscope. Arrows showed that the control oocytes extruded the polar body while the treated oocytes failed. Scale bar, 100 μm. *p < 0.05 vs controls.
Fig 2.
Brusatol treated results in abnormal spindles and chromosome organization defects.
A) Oocytes at MII stage were immunostained with α -tubulin antibody to visualize spindle (green) and counterstained with Hoechst 33342 for chromosomes (blue). Representative confocal images showing the spindle morphology and chromosome alignment in control (a) and Brusatol treated (b-c) oocytes. Arrowheads indicate the abnormal spindle and arrows indicate the misaligned and decondensed chromosomes. B) Quantification of control, Brusatol treated and Brusatol+Nrf2-plasmid treated oocytes with spindle/chromosome defects. Data are expressed as mean percentage ± SD from three independent experiments in which at least 120 oocytes were analyzed. Scale bar, 20 μm. *p < 0.05.
Fig 3.
Brusatol affects oocyte maturation by Nrf2.
A) Western blot showing decline of Nrf2 after Bruastol treatment with actin as a loading control. B) The relative mRNA level of Nrf2 was determined by qRT-PCR in control and Brusatol treated oocytes. mRNA level in control oocytes were set as 1. C) PBS (control group) or Nrf2 CRISPR Activation Plasmid (activation group) was microinjected into GV oocytes, which were arrested for 20 h with milrinone to allow synthesis of new Nrf2 protein. Then the oocytes were collected for Western blot. Results indicated that Nrf2 protein was efficiently overexpressed. D,E) Quantitative analysis of GVBD and Pb1 extrusion in different groups. Data are expressed as mean percentage ± SD from three independent experiments in which at least 80 oocytes were analyzed. *p < 0.05.
Fig 4.
Effects of Brusatol treatment on oxidative stress and mitochondrion.
A) Brusatol reduced the mRNA level of Nrf2 target genes. oocytes were treated with 200 nM of brusatol for 4h, 8h, 14h and subjected to qRT-PCR. B) Representative images of CM-H2DCFDA fluorescence in control, and Brusatol treated oocytes to show the ROS levels. C) Quantitative analysis of fluorescence intensity shown in B. D) Brusatol reduced the mRNA level of mitochondrial related genes. E) To mark the mitochondrion, representative images of mito-tracker fluorescence in control, and Brusatol treated oocytes were taken. F) Quantitative analysis of fluorescence intensity shown in E. Scale bar, 20 μm. *p < 0.05.
Fig 5.
Brusatol reduces cyclin B1 expression in oocytes.
A) The relative mRNA level of cyclin B1 was determined by qRT-PCR in control and Brusatol treated oocytes. mRNA level in control oocytes were set as 1. B) Oocytes at MI stage were immunostained with cyclin B1 antibody (green) and counterstained with Hoechst 33342 for chromosomes (blue). C) Western blot showing decline of cyclin B1 after Bruastol treatment with actin as a loading control. C) Western blot showing increase of cyclin B1 after Nrf2 overexpression with actin as a loading control. Error bars indicate ± sd Scale bar, 20 μm. *p < 0.05.