Fig. 3

Fig. 3

A Rescue of male fertility. Dcst1^d1/d1 males with Dcst1-3xHA Tg insertion and Dcst2^d25/d25 males with Dcst2-3xHA Tg insertion were generated (Fig. S10), and their fertility was rescued [number of plugs: 17 (Dcst1^d1/d1), 25 (Dcst1^d1/d1;Tg), 42 (Dcst2^d25/d25), 15 (Dcst2^d25/d25;Tg)]. The fecundity data in Dcst1^d1/d1 and Dcst2^d25/d25 males is replicated from Fig. 1C. All values are shown as the mean ± SD. B Detection of DCST1 and DCST2 in TGC and sperm. The protein extract of TGC (100 μg) and sperm (6.6 × 10^6 sperm) was used for SDS-PAGE. The HA-tagged DCST1 and HA-tagged DCST2 were detected in TGC and sperm. Total proteins in the membrane were visualized by CBB staining. Triangle marks show the expected molecular size of DCST1 (about 80 kDa) and DCST2 (about 77 kDa). C Localization of DCST2 in sperm. The HA-tagged DCST2 was localized in the anterior acrosome before the acrosome reaction, and then translocated to the equatorial segment in acrosome-reacted sperm (arrows). Wild-type sperm were used as the negative control. PNA was used as a marker for the acrosome reaction. The fluorescence in the sperm tail was non-specific. The scale bars are 25 μm. D Co-IP and western blotting of the interaction between IZUMO1 and DCST1/2. The TGC and sperm lysates from Ctrl (Dcst2^{wt/wt and d25/wt} mice), Dcst1;Tg, and Dcst2;Tg males were incubated with anti-HA tag antibody-conjugated magnetic beads, and then the eluted protein complex was subjected to western blotting. The HA-tagged DCST1 was detected only in the IP product from TGC, and the HA-tagged DCST2 was detected in the IP-product from TGC and sperm. IZUMO1 was not detected in the co-IP products. Red and blue triangle marks show the expected molecular size of DCST1 (about 80 kDa) and DCST2 (about 77 kDa), respectively. E Interaction between DCST1 and DCST2 in HEK293T cells. The protein lysate collected from HEK cells overexpressing Dcst1-3xFLAG and Dcst2-3xHA was incubated with anti-HA tag antibody-conjugated magnetic beads. The FLAG-tagged DCST1 was detected in the eluted protein complex. ADAM1B, a sperm protein that localizes to the sperm surface and is not involved in sperm-egg fusion, was used for negative control.