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SEPTIN12 genetic variants confer susceptibility to teratozoospermia.

Lin YH, Wang YY, Chen HI, Kuo YC, Chiou YW, Lin HH, Wu CM, Hsu CC, Chiang HS, Kuo PL - PLoS ONE (2012)

Bottom Line: The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12.Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage.Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medicine, Fu Jen Catholic University, College of Medicine, Taipei, Taiwan.

ABSTRACT
It is estimated that 10-15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12(+/+)/Septin12(+/-) chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage. Ex vivo experiment showed truncated SEPT12 inhibits filament formation in a dose-dependent manner. This study provides the first causal link between SEPTIN12 genetic variant and male infertility with distinctive sperm pathology. Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

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Effects of truncated - SEPT12 on filament - like formation in NT2D1 cells.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 (**: p<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 (**: p<0.01,*: p<0.05, Student's t test).
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pone-0034011-g004: Effects of truncated - SEPT12 on filament - like formation in NT2D1 cells.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 (**: p<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 (**: p<0.01,*: p<0.05, Student's t test).

Mentions: To determine the functional significance of the truncated SEPT12 protein (SEPT12-del-EGFP) generated from the c.474A/A allele, plasmids encoding SEPT12-EGFP and SEPT12-del-EGFP were transfected into NT2D1 cells, respectively. Over-expressed SEPT12-EGFP formed filaments surrounding the nuclear membrane (Figure 4A.). However, over-expressed SEPT12-del-EGFP aggregated to form a dot- like structure that did not surround the nuclear periphery (Figure 4B and 4E). To test the hypothesis that the mutant protein may influence the function of wild-type protein, we co-transfected cells with SEPT12-del-EGFP and FLAG-SEPT12 expression vectors in different ratios, 1∶ 1, 1∶ 3 and 1∶7. Cells co-transfected with FLAG-SEPT12 and SEPT12-EGFP formed well polymerized filaments around the nuclear membrane (Figure 4C). However, SEPT12-del-EGFP disrupted the filament formation of FLAG-SEPT12 in a dose-dependent manner (Figure 4D and 4F). The finding suggests that truncated SEPT12 lacking the C-terminal may disrupt the polymerization (and filament formation) of the wild-type SEPT12.


SEPTIN12 genetic variants confer susceptibility to teratozoospermia.

Lin YH, Wang YY, Chen HI, Kuo YC, Chiou YW, Lin HH, Wu CM, Hsu CC, Chiang HS, Kuo PL - PLoS ONE (2012)

Effects of truncated - SEPT12 on filament - like formation in NT2D1 cells.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 (**: p<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 (**: p<0.01,*: p<0.05, Student's t test).
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getmorefigures.php?uid=PMC3316533&req=5

pone-0034011-g004: Effects of truncated - SEPT12 on filament - like formation in NT2D1 cells.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 (**: p<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 (**: p<0.01,*: p<0.05, Student's t test).
Mentions: To determine the functional significance of the truncated SEPT12 protein (SEPT12-del-EGFP) generated from the c.474A/A allele, plasmids encoding SEPT12-EGFP and SEPT12-del-EGFP were transfected into NT2D1 cells, respectively. Over-expressed SEPT12-EGFP formed filaments surrounding the nuclear membrane (Figure 4A.). However, over-expressed SEPT12-del-EGFP aggregated to form a dot- like structure that did not surround the nuclear periphery (Figure 4B and 4E). To test the hypothesis that the mutant protein may influence the function of wild-type protein, we co-transfected cells with SEPT12-del-EGFP and FLAG-SEPT12 expression vectors in different ratios, 1∶ 1, 1∶ 3 and 1∶7. Cells co-transfected with FLAG-SEPT12 and SEPT12-EGFP formed well polymerized filaments around the nuclear membrane (Figure 4C). However, SEPT12-del-EGFP disrupted the filament formation of FLAG-SEPT12 in a dose-dependent manner (Figure 4D and 4F). The finding suggests that truncated SEPT12 lacking the C-terminal may disrupt the polymerization (and filament formation) of the wild-type SEPT12.

Bottom Line: The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12.Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage.Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medicine, Fu Jen Catholic University, College of Medicine, Taipei, Taiwan.

ABSTRACT
It is estimated that 10-15% of couples are infertile and male factors account for about half of these cases. With the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring. However, teratozoospermia still remains a big challenge to tackle. Septins belong to a family of cytoskeletal proteins with GTPase activity and are involved in various biological processes e.g. morphogenesis, compartmentalization, apoptosis and cytokinesis. SEPTIN12, identified by c-DNA microarray analysis of infertile men, is exclusively expressed in the post meiotic male germ cells. Septin12(+/+)/Septin12(+/-) chimeric mice have multiple reproductive defects including the presence of immature sperm in the semen, and sperm with bent neck (defect of the annulus) and nuclear DNA damage. These facts make SEPTIN12 a potential sterile gene in humans. In this study, we sequenced the entire coding region of SEPTIN12 in infertile men (n = 160) and fertile controls (n = 200) and identified ten variants. Among them is the c.474 G>A variant within exon 5 that encodes part of the GTP binding domain. The variant creates a novel splice donor site that causes skipping of a portion of exon 5, resulting in a truncated protein lacking the C-terminal half of SEPTIN12. Most individuals homozygous for the c.474 A allele had teratozoospermia (abnormal sperm <14%) and their sperm showed bent tail and de-condensed nucleus with significant DNA damage. Ex vivo experiment showed truncated SEPT12 inhibits filament formation in a dose-dependent manner. This study provides the first causal link between SEPTIN12 genetic variant and male infertility with distinctive sperm pathology. Our finding also suggests vital roles of SEPT12 in sperm nuclear integrity and tail development.

Show MeSH
Related in: MedlinePlus