Sperm binding capability and ZP2 cleavage status.

(A, B) Eggs and two-cell embryos from control and melamine fed mice were incubated with capacitated sperm for 1 hr. After washing with a wide-bore pipette to remove all but two to six sperm on normal two-cell embryos (negative control), eggs and embryos with sperm were fixed and stained with Hoechst 33342. Scale bar, 20μm. (C) The number of sperm binding to the surface of zona pellucida surrounding eggs. There was no significant difference between control (n = 10) and high-dose (n = 8) groups. (D) Western blot analysis of ZP2 cleavage status in eggs and two-cell embryos using M2c.2 antibody that recognizes the C-terminal domain of ZP2. The size of intact ZP2 is 120 kD, and the size of cleaved C-terminal fragment of ZP2 is 90 kD.