Expression patterns of SEPT12 human male germ cells with the c.474G/G (wild) and c.474A/A genotypes.

<p>(A.) Detection of SEPT12 signals during human spermiogenesis. (a.–a′): Round Spermatids (RS), (b.–b′) Elongating Spermatids (ES) and Mature Sperm (MS). Left: SEPT12 signal (green); Right: merge of SEPT12 (green) and DAPI (light blue) signals. (B.) Varied type of spermatozoa isolated from cases with the c.474A/A genotype. Left: SEPT12 signal (green); Right: merge of SEPT12 (green) and DAPI (light blue) signals (Magnification: ×1,000).</p

Identification of novel variants in the SEPTIN12 gene.

<p>Genomic structure of the <i>SEPTIN12</i> gene and positions of the ten SNPs. Open bars indicate exons. The ATG start site is located at exon 2. Exon 3 to exon 8 encodes the GTP -Binding Domain of SETIN12. (B.–C.) Electropherograms showing DNA sequences. Lower panels show the variant (c.474C→A, Left; c.494T→A, Right) sequences, whereas the upper panels show the wild-type (normal) sequences. Red stars indicate locations of the variants.</p

Abnormal morphology of spermatozoa from a case with c.474A/A.

<p>(A.) Motile sperm organelle morphology examination (MSOME) for sperm cells using a high-magnification inverted microscope (magnification was ×8400). (a.) sperm from a fertile control with c.474C/C; (b.–d.) sperm from an infertile man with c.474A/A. Sperm with bent-tail (b.), neck(c.) and round head (d.). (B.) IFA assay for sperm from a fertile control with c.474C/C (a.) and an infertile man with c.474A/A (b.). DAPI: blue; mito-tracker: red. (Magnification: ×400).</p

Nuclear DNA damage in the spermatozoa of infertile men who carried c.474A/A.

<p>(A.–C.) The spermatozoa were stained with AO (A.), TB (B.) and AB (C.) dyes. (A.) The spermatozoa with normal (green) or abnormal nucleus (yellow). (B.–C.) Spermatozoa with normal (light blue) or abnormal (dark blue) nucleus. (Magnification: ×1,000). (D.) Quantification of the percentage of AO-, AB- or TB- stained spermatozoa. At least 100 spermatozoa cells were counted in each case (*: <i>p</i><0.05; Mann-Whitney test).</p

Effects of truncated - SEPT12 on filament - like formation in NT2D1 cells.

<p>Immuno-fluorescence assay (IFA) shows wild- SEPT12-EGFP (SEPT12-EGFP) (A.) or truncated- SEPT12-EGFP (SEPT12-del-EGFP) (B.) forms filament - like or dot- like structure, respectively. (A.–B.) Merged pictures for staining with anti-EGFP antibody (green) and DAPI (light blue). The results of co-expressed wild- SEPT12-EGFP (SEPT12-EGFP) (C.) or truncated- SEPT12-EGFP (SEPT12-del-EGFP) (D.) with wild- FLAG-SEPT12 (FLAG-SEPT12) in cells are presented in (C.) and (D.), respectively. Signals from EGFP protein (green), anti-FLAG antibody (red) and DAPI (Light blue) signals are merged in (C.) and (D.). (A.–D.) Arrows indicated filament-like structure; Arrow head indicated dot-like structure. Magnification: ×400 in A–D. (E.–F) Quantification of the percentage of filament- like structures in transfected cells. The height of the boxes represents the mean of value obtained from four independent experiments. At least 100 transfected cells were counted in each experiment (**: <i>p</i><0.01, Student's t test). (F.) Dosage- dependent inhibition of filament-like formation by the truncated SEPT12 protein. Plasmids encoding FLAG-tagged wild-type SEPTIN12 were mixed with various amounts of plasmids encoding EGFP-tagged wild- or truncated SEPTIN12, then the mixtures were transfected into NT2D1 cells (**: <i>p</i><0.01,*: <i>p</i><0.05, Student's t test).</p

SEPTIN12 allele frequencies in infertile men and control subjects.

<p>Nucleotide numbering indicates cDNA numbering with 1+ corresponding to the A of the ATG translation initiation codon in the reference cDNA sequence of <i>SEPTIN12</i> (<b>NM_144605.3</b>).</p

Spermatozoa from c.474A/A patients with abnormal head shape.

<p>(A.–B.) TEM images of sperm isolated from a fertile control (A.) and an infertile mam with c.474A/A (B). The latter shows de-condensed chromatin. Arrows indicate the nucleus; arrow heads indicate the axonemal 9+2 structures (Magnification: ×10,000). (C.–E.) Top-view AFM images confirm abnormal morphology in sperm head. Sperm of a control subject (C.). Sperm of an infertile man with c.474A/A have a narrow head (D.) or a de-condensed nucleus (E.). Three-dimensional images are displayed in the bottom (C′.–E′.).</p

Effects of the c.474G→A variant on the splicing process.

<p>(A.) The c.474 G→A SNP induces alternative splicing <i>ex vivo</i>. Left panel: PCR fragments containing exon 5 (E5, back and gray box), intron 5 (within splice donor site “gt” and splice acceptor site “ag”) and exon 6 (E6, black box) with c.474 G or c.474A were constructed into a vector, respectively. Middle panel: products of RT–PCR are shown on an agarose gel. Control: <i>GAPDH</i> Two transcripts (wild-type and alternatively spliced)are produced by the minigene with c.474A. Right panel: schematic depiction of the RT–PCR products. Wild-type transcript: using the original splice donor and acceptor site. Alternatively spliced transcript: using the novel splice donor induced by c.474G→A and the original splice acceptor site. (B.) Sequences of the wild-type (upper panel) and alternatively spliced (lower panel) products from the minigene containing c.474A. The alternatively spliced transcript induces a novel splice donor site, which results in skipping of partial exon 5 (with loss of 41 bp), and also created a premature stop codon in exon 6.</p