Figure 3
Characterisation of Odf2+/− males. (A) (Top) Agouti coat-coloured pups produced by intracytoplasmic headneck injection. (Bottom) Genotyping. +/+: Wild-type (180). +/−: Heterozygous Odf2+/− (180 and 280). (B) Pup production of heterozygous Odf2+/− (+/−) females is normal. (C) Pup production of heterozygous Odf2+/− (+/−) males is low (0) compared to that of wild-type (+/+) (9.2). (D) (Left) Western blotting. (Top) Reduced amount of the 75 kDa Odf2 protein in Odf2+/− (+/−) compared to that in Odf2+/+ (+/+). Full-length membranes (CBB staining and western blotting); all data are presented in Fig. S4. (Bottom) β-Tubulin; internal control. The same amount of protein (0.5 μg/lane) was loaded. (Right) The reduction is quantitatively significant (*P < 0.05). The immunostaining intensity obtained by western blotting (Left image) was subjected to analysis with Image Lab Software in a Bio-Rad ChemiDoc XRS+ (molecular imager). The mean ± SEM is 243 × 104 ± 16 × 104 in Odf2+/− versus 400 × 104 ± 28 × 104 in Odf2+/+. (E) The display shows the western blots for the midpiece-related proteins cropped from the same or different membranes. Full-length membranes (CBB staining and western blotting) for all samples are presented in Fig. S5. No marked difference in the midpiece-related proteins between the wild-type (+/+) (Left) and heterozygotes (+/−) (Right). β-Tubulin was used as an internal control. The same amount of protein per lane was loaded for Odf1 (1 μg), Speriolin (3 μg), Tektin4 (16 μg), AK-1 (16 μg), Septin7 (6 μg), PHGPx (16 μg), GAPDH (16 μg) and β-Tubulin (16 μg). (F) Classification of sperm morphology (brightfield). (G) Percentages (%) of abnormal spermatozoa (Left; large pie chart) and headnecks among abnormal spermatozoa (Right; small pie chart) are high in Odf2+/− (+/−) (99.7% and 99.4%, respectively) compared to those in Odf2+/+ (+/+) (2.5% and 0%, respectively). The total number of samples examined is written at the bottom of the pie charts. (H) Representative images at low magnification (brightfield) of Odf2+/+ (+/+) and Odf2+/− (+/−) spermatozoa. Numerals (1–12) in each figure are sperm number. The proportion of headnecks shown in these figures is well in accord with that of G (Right).
