Immunofluorescence Analysis of Zygote

Ten hours after the ICSI, the zona pellucidae of surviving oocytes were removed using acetic tyrode solution, and then the naked oocytes were fixed for 30 min at 25°C in 4% (w/v) paraformaldehyde (PFA). The fixed oocytes were washed three times in phosphate-buffered saline (PBS)–polyvinyl alcohol (0.1 mg/ml; Sigma-Aldrich, St. Louis, MO) for 10 min and stored overnight at 4°C in PBS supplemented with 1% (w/v) bovine serum albumin (BSA/PBS; Sigma-Aldrich) and 0.1% (v/v) Triton X-100 (Nacalai Tesque Inc., Kyoto, Japan). The following procedures are previously described in (30 (link)). The primary antibodies used were an anti–phospho-H2AX (Ser¹³⁹) rabbit polyclonal antibody (1:500; Millipore-Merck, Darmstadt, Germany) and an anti-histone H3 (dimethyl K9) mouse monoclonal antibody (1:500, Abcam, Cambridge, UK). The secondary antibodies used were Alexa Fluor 488–labeled goat anti-mouse immunoglobulin G (IgG; 1:500, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568–labeled goat anti-rabbit IgG (1:500 dilution; Molecular Probes). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI; 2 μg/ml; Molecular Probes). The brightness of the whole male pronucleus was measured using ImageJ and was then subtracted from the brightness of the zygote cytoplasm.