Figure 3. Identification of new gamete fusion-related factors dendrocyte expressed seven transmembrane protein domain-containing 1 (DCST1) and dendrocyte expressed seven transmembrane protein domain-containing 2 (DCST2).
(A) Accumulation of many acrosome-reacted (AR) IZUMO1 positive spermatozoa (red) in the perivitelline space of eggs recovered from the oviducts of females 8 hr after coitus with a Dcst1/2-/- male. Scale bar 10 µm. (B) Gamete fusion assay of Dcst1/2-disrupted spermatozoa. Gamete fusion was visualized with Hoechst 33342 dye transfer. The numbers of fused spermatozoa per oocyte from four independent experiments are shown. The total numbers of oocytes examined in Dcst1/2+/- and Dcst1/2-/- were 77 and 80, respectively (left graph). The broken lines indicate the average. The arrows and the circles in the representative photo show fused spermatozoa and meiosis metaphase II chromosomes, respectively (right picture). Scale bar 10 µm. (C) Localization of oocyte CD9 and JUNO upon Dcst1/2-/- sperm attachment. Sperm–oocyte interaction was visualized by staining with 0.25 µg ml−1 TH6–Alexa647 (JUNO: red), 0.5 µg ml−1 MZ3–FITC (CD9: green) and 0.5 µg ml−1 Mab125–Alexa546 (IZUMO1: blue). The eggs were fixed 30 min after insemination of Dcst1/2-/- spermatozoa. The arrowheads indicate the sites where JUNO and cluster of differentiation 9 (CD9) were concentrated in response to the AR spermatozoa (IZUMO1 positive: blue) bound to the oolemma. Scale bar 10 µm. (D) In vitro fertilization assay using Dcst1/2+/-, Dcst1/2-/-, Dcst1+/-, Dcst1-/-, Dcst2+/-, Dcst2-/-, Dcst1/2-/-Dcst1-TG, Dcst1/2-/-Dcst2-TG, and Dcst1/2-/-Dcst1/2-TG spermatozoa (n = 5, 5, 6, 7, 8, 8, 6, 6, and 6, respectively). The fertilization rate was evaluated at the two-cell stage embryo. The numbers in parentheses indicate the numbers of oocytes used. The error bars represent s.e.m. ***p<0.001 (paired two-tailed Student’s t-test, compared with each heterozygous mutants). (E) Fecundity of DCST1/2-related deficient male and female mice. The numbers in parentheses indicate the numbers of mating pairs. Regarding Dcst1/2-/-, Dcst1-/-, Dcst2-/-, Dcst1/2-/-Dcst1-TG, and Dcst1/2-/-Dcst2-TG males, the numbers of vaginal plug formations were counted. The error bars represent s.e.m. (F) Western blot analysis of various types of mouse lines with 1 µg ml−1 Mab18 (IZUMO1) and 1:500 SPACA6 anti-serum. Each lane was equally applied with 20 µg sperm lysates. BASIGIN was used as a loading control.