Fig. 2

Fig. 2

A Binding ability. Dcst1 KO and Dcst2 KO sperm could bind to the oolemma after 30 min of incubation. The scale bars are 50 μm. B Detection of IZUMO1. The band signals of IZUMO1 in TGC and sperm of Dcst1^d1/d1 and Dcst2^d25/d25 male mice were comparable to the control wild-type sperm. SLC2A3, one of proteins in sperm tail, was used as the loading control. C, D Acrosome status of binding sperm. Live sperm bound to the oolemma were stained with the IZUMO1 antibody, and IZUMO1 only in the acrosome reacted (AR) sperm was detected. The AR-sperm of both Dcst mutants could bind to the oolemma, and their numbers were higher than the control heterozygous sperm [3.27 ± 2.31 (Ctrl, 93 eggs), 7.34 ± 5.09 (Dcst1^d1/d1, 68 eggs), 4.74 ± 2.93 (Dcst2^d25/d25, 89 eggs)]. **p < 0.01. The scale bars are 25 μm. E, F Fusion ability. The ZP-free eggs pre-stained Hoechst 33342 were used for sperm-egg fusion assay. Hoechst 33342 signal transferred to control sperm heads, indicating that sperm fused with eggs (panel E, arrow). However, Dcst1 KO and Dcst2 KO sperm rarely fused with eggs [fused sperm/egg: 1.52 ± 0.35 (Ctrl, 113 eggs), 0.04 ± 0.05 (Dcst1^d1/d1, 73 eggs), 0 (Dcst2^d25/d25, 73 eggs)]. The scale bars are 25 μm. All values are shown as the mean ± SD.