Fig. 7: Blockage of GalNAc on the UTJ and ZP surface decreased the sperm number bound to the UTJ and ZP.

a Detection of GalNAc in the UTJ. By the lectin histochemistry with Rhodamine-labeled Dolichos biflorus agglutinin (DBA) which recognizes the terminal α-GalNAc, DBA bound to the epithelium in UTJ. The Rhodamine-labeled DBA preabsorbed with GalNAc was used as the N.C. The area dotted by the white color was magnified (right panels). The data reproducibility was checked by three biological replicates. b Detection of GalNAc in the ZP. By live cell staining with Rhodamine-labeled DBA, DBA bound to the surface of ZP. The Rhodamine-labeled DBA preabsorbed with GalNAc was used as the N.C. The data reproducibility was checked by three biological replicates. c Sperm-UTJ binding assay. To evaluate the possibility of sperm-UTJ binding through GalNAc residues on the UTJ, the UTJ was incubated with DBA or DBA preabsorbed with GalNAc. After washing with TYH medium, the UTJ was inseminated with sperm pre-stained with Hoechst 33342. After fixation and washing, the sperm bound to the UTJ were observed. The Hoechst signal (the display changed to grayscale) in the area dotted by the black color was magnified (right panels). The data reproducibility was checked by four biological replicates. d Sperm-ZP binding assay. To evaluate the possibility of sperm-ZP binding through GalNAc on the ZP, eggs were incubated with DBA or DBA preabsorbed with GalNAc (see Supplementary Fig. 6). The eggs were washed with TYH medium and then inseminated with sperm for 30 min. After fixation and washing, the eggs were observed (left panel), and the sperm number bound to ZP was counted (right panel) (DBA: 6.8 ± 5.3, DBA + GalNAc: 13.4 ± 7.5) (Mann–Whitney test, p < 0.0001). Center line within the box: median, whiskers in box-and-whiskers plots: minimum to maximum, ****p < 0.001.