FIGURE 3.

Impaired sperm binding to the zona pellucida in Calr3^−/− mice. A, wild-type sperm successfully bound to the egg zona pellucida, but Calr3^−/− sperm failed to adhere despite frequent collisions (supplemental Movies S1 and S2). B, Calr3^−/− sperm were not able to fertilize eggs in vitro. The average fertilization rate from three independent experiments is presented. The total number of eggs examined is indicated in parentheses. IVF, in vitro fertilization. C and D, zona–free eggs preloaded with Hoechst 33342 were incubated with +/+ or Calr3^−/− sperm, and fused sperm were observed under a fluorescence microscope. Arrows indicate MII chromosomes, and arrowheads indicate fused sperm (C). The average fusion rate (fused eggs/examined eggs) from three independent experiments is presented in D. The number of eggs examined is indicated in parentheses. E–G, successful fertilization was observed with Calr3^−/− sperm after partial zona dissection and in vitro fertilization. Arrowheads indicate the slit made by partial zona dissection (E). The offspring of Calr3^−/− males obtained after partial zona dissection, in vitro fertilization, and embryo transfer are shown in F. The average birth rate (pups/transplanted eggs) after transferring embryos subjected to partial zona dissection and in vitro fertilization (n = 3) is shown in G.