Efficient knockdown of RAD18 in spermatocytes from Rad18 KD mice, and increased frequency of XY asynapsis. (A,B) Double immunostaining of pachytene spermatocyte nuclei of control (A) and Rad18 KD mice (B) with anti-SYCP3 (red) and anti-RAD18 (green). The insert shows a larger magnification of the area containing the XY body. Arrows indicate the pseudoautosomal synapsed region (A) and unsynapsed region (B). (C) RAD18 intensity on the XY body was measured with ImageJ software. The intensity of RAD18 in control nuclei was set at 1.0. Error bars indicate s.e.m. **P<0.01 (Mann–Whitney U-test). (D) The percentage of unsynapsed X and Y chromosomes in mid-pachytene of 4-week-old and 19-week-old mice. **P<0.01 (Chi-squared test). (E,F) Immunostaining of Rad18 KD pachytene spermatocyte nuclei with anti-SYCP3 (red). The insert shows a larger magnification of the area containing the XY body. X and Y chromosomes are indicated in the enlarged pictures. (G) 4-week-old wild-type and Hr6b KO mice. (C,D,G) Blue and red bars indicate control (ctr) and Rad18 KD, respectively.