Delayed PMBP establishment by Plcz1 deletion is a cause of polyspermy. (a) Representative fluorescence plots for R-GECO (intracellular Ca2+; gray) and LCA–FITC staining (CR; black) of oocytes after insemination. Black arrowheads highlight Ca2+ spikes that induced the CR, while white arrowheads indicate Ca2+ spikes that failed to initiate the CR. (b) ZP2 immunoblot of oocytes after insemination. Intact ZP2 and the cleaved C-terminal fragments of ZP2 were 120 kD and 90 kD, respectively. A full–length blot is presented in Supplementary Figure 7a. (c) PMBP establishment estimated with the number of fused sperm of in vitro fertilized oocytes. The data reflect the means of separate experiments for each genotype. (d) Oocytes collected from oviduct of Astl heterozygous or KO females mated with WT male. Bright field observation (upper) and nuclear staining (lower) using Hoechst 33342 (blue) at 12 h post coitum are indicated. Oocytes from Astl KO females were denuded to prevent spermatozoa within the perivitelline space (arrowheads) interfering with pronuclei counting. The numbers of 2PN oocytes/total oocytes from five females for each genotype are indicated. No KO oocyte exhibited polyspermy. Scale bar = 50 μm.