Immunofluorescence Analysis of Mouse Oocytes

The immunofluorescence staining of mouse oocytes was performed as previously described [20 (link)]. Briefly, the spindle and chromosome were stained using anti-mouse β-tubulin antibody (CST, Boston, MA, USA) and Hoechst 33258 (Servicebio, Wuhan, China), respectively. DNA damage levels were evaluated using anti-rabbit phospho-histone H2AX antibody (CST, Boston, MA, USA). For evaluation of intra-oocyte ROS and MMP, the oocytes were stained with the 2′,7′- dichlorodihydro- fluorescein diacetate (DCHF-DA) (Sigma, St. Louis, MO, USA) and MMP detection (JC-1) kit (Beyotime Biotechnology Research Institute, China), respectively. The stained oocytes were observed and photographed under a fluorescence microscope (Carl Zeiss, Batenwerburg, Germany) using fixed microscopic parameters. Image J software (NIH, Bethesda, MD, USA) were applied to measure the fluorescence intensities.