Male <i>Tysnd1</i><sup>−/−</sup> mice are infertile.

<p>A. Semi-thin (8–10 micron) testes sections of 20 weeks old <i>Tysnd1</i><sup>+/+</sup> and <i>Tysnd1<sup>−/−</sup></i> mice were stained with hematoxylin-eosin. Abnormal, round sperm heads are visible in the seminiferous tubules of <i>Tysnd1</i><sup>−/−</sup> mice. B. Abnormal morphology of <i>Tysnd1</i><sup>−/−</sup> sperms. Epididymal sperms of 15 weeks-old <i>Tysnd1</i><sup>+/+</sup> and <i>Tysnd1</i><sup>−/−</sup> mice were stained with mitochondrial stains (MitoFluor Red) and nuclear staining (DAPI). Scale bar = 20 µm. C. Percentage of sperms showing normal morphology in <i>Tysnd1</i><sup>+/+</sup> (n = 4), <i>Tysnd1</i><sup>+/−</sup> (n = 4) and <i>Tysnd1</i><sup>−/−</sup> (n = 5) mice. Each error bar represents the mean ± SE. D. Anti-MN9 antibody immunostaining and Hoechst nuclear staining of epididymal sperms isolated from a 20 weeks old <i>Tysnd1</i><sup>−/−</sup> mouse (red: acrosome and blue: nucleus). Arrow heads and arrows indicate abnormal round-headed sperms and normal sperms, respectively. Scale bar = 5 µm. E. Acrosomes of a semi-thin testis section from a 10 weeks old <i>Tysnd1</i><sup>−/−</sup> and a heterozygous control mouse were stained with PNA-FITC (green) and Hoechst nuclear stain (blue). F. EM image of a <i>Tysnd1</i><sup>−/−</sup> caudal epididymal sperm. The round-headed sperm lacks the acrosome and shows an abnormal mitochondrial sheath (M) around the nucleus (N). Scale bar = 1 µm. G. EM image showing normal spermatogenesis in <i>Tysnd1</i><sup>+/−</sup> male mice. S7: step 7 round spermatid; S16: step 16 elongated spermatid. Acrosomes (A) are normally formed. Scale bar = 2 µm. H. EM image of <i>Tysnd1</i><sup>−/−</sup> elongated spermatid. S16: step 16 spermatid. In some spermatids the acrosome (*) is detached from the nucleus (N). Scale bar = 2 µm.</p>

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