Figure 1.
Antibody specificity and native VDAC2 in human spermatozoa analyzed by western blot.
A: recombinant VDAC1 (lane 1), VDAC2 (lane 2) and VDAC3 (lane 3) proteins were probed with anti-VDAC2 antibody. B: the hydrophobic membrane protein extracts from human spermatozoa were separated by SDS-PAGE and probed with anti-VDAC2 antibody (lane 1) or the same antibody pre-absorbed with an excess of recombinant VDAC2 protein. The position of molecular weight standards (kDa) is indicated on the left.
Figure 2.
FITC-PSA staining of human spermatozoa in different treatment groups.
A–C: spermatozoa were separated and stained immediately. D–F: spermatozoa were incubated with anti-VDAC2 antibody. G–I: spermatozoa were incubated with normal mouse IgG. J–L: spermatozoa were incubated without any antibody. A, D, G and J: phase-contrast images; B, E, H and K: immunofluorescent images; C, F, I and L: merged images. Magnification was ×1000.
Figure 3.
Effect of anti-VDAC2 antibody on acrosomal integrity.
A: spermatozoa were incubated with normal mouse IgG or anti-VDAC2antibody at different dilutions and counted according to the different incubation time. B: spermatozoa were incubated for the different time and counted according to different treatments. At least 200 spermatozoa were counted in each sample. Data were shown as mean ± SEM of six experiments; *, P < 0.05 versus control.
Figure 4.
FITC-PSA staining of human spermatozoa in different treatment groups.
A–C: spermatozoa were loaded with anti-VDAC2 antibody and induced by A23187. D–F: spermatozoa were loaded with normal mouse IgG and induced by A23187. G–I: spermatozoa were not loaded with any antibody and induced by A23187. A, D and G: phase-contrast images; B, E and H: immunofluorescent images; C, F and I: merged images. Magnification was ×1000.
Figure 5.
Effect of anti-VDAC2 antibody on acrosome reaction.
A: spermatozoa were incubated for 2 h, loaded with normal mouse IgG or anti-VDAC2antibody at different dilutions, and counted with or without A23187. B: spermatozoa were incubated for 4 h, loaded with normal mouse IgG or anti-VDAC2antibody at different dilutions, and counted with or without A23187. At least 200 spermatozoa were counted in each sample. Data were shown as mean ± SEM of six experiments.
Figure 6.
Effect of anti-VDAC2 antibody on A23187-induced Ca2+ influx.
Spermatozoa were incubated for 2 h, then loaded with anti-VDAC2 antibody (yellow), normal mouse IgG (blue), or without any antibody (red), and finally stimulated by A23187. The changes of fluorescence intensity reflecting intracellular Ca2+ concentration were recorded by the Microplate Reader.