CRL-3518 Search Results


human dnah17 amino acids 3518 3817  (ATCC)
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ATCC human dnah17 amino acids 3518 3817
A <t>DNAH17</t> missense variant in a consanguineous Pakistani family with asthenozoospermia. (A) Pedigree of the consanguineous family with three asthenozoospermia patients (IV:1, IV:2, and IV:3). Arrows point to the two individuals for whom WES was performed. Slashes denote deceased family members, and the double horizontal lines represent consanguineous marriage. ASZ, asthenozoospermia. (B) Chromatograms of the DNAH17 missense mutation (g.G78136A) in genomic DNA from all the available family members. F, female; M, male. (C) The DNAH17 mutation occurs in exon 35 and causes a G-to-A substitution at cDNA (NCBI reference sequence no. NM_173628 ) nucleotide position 5408, replacing cysteine (C) with tyrosine (Y) at amino acid 1803 in the DNAH17 protein (UniProt accession no. Q9UFH2 ). (D) Sequence alignment shows conservation of the affected amino acid (cysteine) across different organisms. Arrowheads, the mutation site; WT, the wild-type allele; MT, the mutant allele; UTR, untranslated region.
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A DNAH17 missense variant in a consanguineous Pakistani family with asthenozoospermia. (A) Pedigree of the consanguineous family with three asthenozoospermia patients (IV:1, IV:2, and IV:3). Arrows point to the two individuals for whom WES was performed. Slashes denote deceased family members, and the double horizontal lines represent consanguineous marriage. ASZ, asthenozoospermia. (B) Chromatograms of the DNAH17 missense mutation (g.G78136A) in genomic DNA from all the available family members. F, female; M, male. (C) The DNAH17 mutation occurs in exon 35 and causes a G-to-A substitution at cDNA (NCBI reference sequence no. NM_173628 ) nucleotide position 5408, replacing cysteine (C) with tyrosine (Y) at amino acid 1803 in the DNAH17 protein (UniProt accession no. Q9UFH2 ). (D) Sequence alignment shows conservation of the affected amino acid (cysteine) across different organisms. Arrowheads, the mutation site; WT, the wild-type allele; MT, the mutant allele; UTR, untranslated region.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: A DNAH17 missense variant in a consanguineous Pakistani family with asthenozoospermia. (A) Pedigree of the consanguineous family with three asthenozoospermia patients (IV:1, IV:2, and IV:3). Arrows point to the two individuals for whom WES was performed. Slashes denote deceased family members, and the double horizontal lines represent consanguineous marriage. ASZ, asthenozoospermia. (B) Chromatograms of the DNAH17 missense mutation (g.G78136A) in genomic DNA from all the available family members. F, female; M, male. (C) The DNAH17 mutation occurs in exon 35 and causes a G-to-A substitution at cDNA (NCBI reference sequence no. NM_173628 ) nucleotide position 5408, replacing cysteine (C) with tyrosine (Y) at amino acid 1803 in the DNAH17 protein (UniProt accession no. Q9UFH2 ). (D) Sequence alignment shows conservation of the affected amino acid (cysteine) across different organisms. Arrowheads, the mutation site; WT, the wild-type allele; MT, the mutant allele; UTR, untranslated region.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Variant Assay, Mutagenesis, Sequencing

Alignment of human and mouse DNAH17 protein sequences showing that 91% of amino acids are identical. Residues that are identical between human and mouse DNAH17 appear in red and as uppercase letters in the consensus line. Residues highly similar between human and mouse DNAH17 are indicated by red symbols (!, any one of I and V; $, any one of L and M; %, any one of F and Y; #, any one of N, D, Q, E, B, and Z). Unconserved residues are written in blue or as asterisks in the consensus line. Blue lines highlight the epitope (amino acids 3502–3801 for mouse DNAH17, corresponding to amino acids 3518–3817 in human DNAH17) for antibody generation. The alignment was performed using the online software MultAlin ( http://multalin.toulouse.inra.fr/multalin/multalin.html ).

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Alignment of human and mouse DNAH17 protein sequences showing that 91% of amino acids are identical. Residues that are identical between human and mouse DNAH17 appear in red and as uppercase letters in the consensus line. Residues highly similar between human and mouse DNAH17 are indicated by red symbols (!, any one of I and V; $, any one of L and M; %, any one of F and Y; #, any one of N, D, Q, E, B, and Z). Unconserved residues are written in blue or as asterisks in the consensus line. Blue lines highlight the epitope (amino acids 3502–3801 for mouse DNAH17, corresponding to amino acids 3518–3817 in human DNAH17) for antibody generation. The alignment was performed using the online software MultAlin ( http://multalin.toulouse.inra.fr/multalin/multalin.html ).

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Software

Validation of the anti-DNAH17 antibody in transfected cells and Dnah17 knockout mice. (A and B) Immunoblotting (A) and IF staining (B) of HEK293T cells overexpressing the epitope-corresponding peptides from human DNAH17 (amino acids 3518–3817), mouse DNAH17 (amino acids 3502–3801), human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839) with an N-terminal FLAG tag. For immunoblotting, untransfected cells were used as negative control. Two independent experiments were performed. Scale bars represent 10 µm. (C) Genomic DNA sequencing chromatograms showing the one-nucleotide-deletion mutation (g.971_971del) in Dnah17 −/− mice. Arrowheads indicate the mutation site. WT, the wild-type allele. MUT, the mutant allele. (D) Immunoblotting using the anti-DNAH17 antibody detected a specific band at the predicted size of DNAH17 (512 kD), as indicated by the arrowhead, in spermatozoa from Dnah17 +/− mice, but not in spermatozoa from Dnah17 −/− mice. ODF2 (a marker of outer dense fibers) was used as the loading control. (E) Representative images of spermatozoa from Dnah17 +/− and Dnah17 −/− mice stained for DNAH17 and α-tubulin, a marker for sperm flagellum. Scale bars represent 10 µm. (F) Sperm count per epididymis in Dnah17 −/− mice. (G) Quantification of the spermatozoa with normal morphology. (H) Representative images of spermatozoa after Papanicolaou staining showing absent (i), short (ii), coiled (iii), bent (iv), and irregular-caliber (v) flagella in Dnah17 −/− mice. Scale bars represent 5 µm. (I) Frequencies of sperm flagella that were morphologically normal, absent, short, coiled, bent, or of irregular caliber. Each spermatozoon was classified as only one type of flagellar morphology according to its major abnormality. (J) Representative TEM micrographs showing cross sections of proximal (upper panel) and distal (lower panel) regions of sperm flagella from Dnah17 +/− and Dnah17 −/− mice. Scale bars represent 200 nm. (K) Immunoblotting using the anti-DNAH17 antibody detected a specific band of predicted size (510 kD for human DNAH17 and 512 kD for mouse DNAH17), as indicated by the arrowhead, in testicular lysates from adult WT mice (mTestis) or fertile men (hTestis). GAPDH was used as the loading control (GAPDH was predicted to be 35.8 kD in mouse and 36.1 kD in human). For A–K, at least two independent experiments were performed. (F, G, and I) Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. N, number of mice examined; n, number of spermatozoa examined.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Validation of the anti-DNAH17 antibody in transfected cells and Dnah17 knockout mice. (A and B) Immunoblotting (A) and IF staining (B) of HEK293T cells overexpressing the epitope-corresponding peptides from human DNAH17 (amino acids 3518–3817), mouse DNAH17 (amino acids 3502–3801), human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839) with an N-terminal FLAG tag. For immunoblotting, untransfected cells were used as negative control. Two independent experiments were performed. Scale bars represent 10 µm. (C) Genomic DNA sequencing chromatograms showing the one-nucleotide-deletion mutation (g.971_971del) in Dnah17 −/− mice. Arrowheads indicate the mutation site. WT, the wild-type allele. MUT, the mutant allele. (D) Immunoblotting using the anti-DNAH17 antibody detected a specific band at the predicted size of DNAH17 (512 kD), as indicated by the arrowhead, in spermatozoa from Dnah17 +/− mice, but not in spermatozoa from Dnah17 −/− mice. ODF2 (a marker of outer dense fibers) was used as the loading control. (E) Representative images of spermatozoa from Dnah17 +/− and Dnah17 −/− mice stained for DNAH17 and α-tubulin, a marker for sperm flagellum. Scale bars represent 10 µm. (F) Sperm count per epididymis in Dnah17 −/− mice. (G) Quantification of the spermatozoa with normal morphology. (H) Representative images of spermatozoa after Papanicolaou staining showing absent (i), short (ii), coiled (iii), bent (iv), and irregular-caliber (v) flagella in Dnah17 −/− mice. Scale bars represent 5 µm. (I) Frequencies of sperm flagella that were morphologically normal, absent, short, coiled, bent, or of irregular caliber. Each spermatozoon was classified as only one type of flagellar morphology according to its major abnormality. (J) Representative TEM micrographs showing cross sections of proximal (upper panel) and distal (lower panel) regions of sperm flagella from Dnah17 +/− and Dnah17 −/− mice. Scale bars represent 200 nm. (K) Immunoblotting using the anti-DNAH17 antibody detected a specific band of predicted size (510 kD for human DNAH17 and 512 kD for mouse DNAH17), as indicated by the arrowhead, in testicular lysates from adult WT mice (mTestis) or fertile men (hTestis). GAPDH was used as the loading control (GAPDH was predicted to be 35.8 kD in mouse and 36.1 kD in human). For A–K, at least two independent experiments were performed. (F, G, and I) Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t test. N, number of mice examined; n, number of spermatozoa examined.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Transfection, Knock-Out, Western Blot, Staining, FLAG-tag, Negative Control, DNA Sequencing, Mutagenesis, Marker

Expression and localization of DNAH17 in humans and mice. (A and B) Representative images of mouse (A) and human (B) respiratory cilia stained for α-tubulin (a marker for the ciliary axoneme) and rabbit IgG (negative control, upper panel) or DNAH17 (lower panel). Scale bars represent 10 µm. (C) Quantitative real-time PCR analysis of Dnah17 mRNA expression in adult mouse tissues. Actb was used as an internal control. (D) Immunoblotting analysis of DNAH17 protein in different tissues from adult mice. GAPDH was used as the loading control. (E) Immunoblotting with sperm lysates from WT mice and epididymal lysates from Rpl10l +/− and Rpl10l −/− mice using the anti-DNAH17 antibody. Lamin B1 was used as the loading control. (F) Representative images of testicular tubules stained with anti-DNAH17 antibody and Hoechst showing that DNAH17 is localized in the cytoplasm and flagella of step 11–16 spermatids. Scale bars represent 50 µm. (G) Immunoblotting with lysates of human cell lines HepG2 (from liver), HEK293T (from embryonic kidney), HCT116 (from colon), A549 (from alveolar basal epithelia), U2OS (from bone), HeLa (from cervix), and adult human testes (hTestis) using the anti-DNAH17 antibody. GAPDH was used as the loading control. (H) Immunohistochemistry using the anti-DNAH17 antibody on adult human testicular sections with normal spermatogenesis. Rabbit IgG (left panel) was used as a negative control. Scale bars represent 50 µm. (I) Representative images of spermatozoa from fertile men (controls) stained with anti-DNAH17 antibody, anti–α-tubulin antibody, and Hoechst. Scale bar represents 10 µm. For A–I, three independent experiments were performed.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Expression and localization of DNAH17 in humans and mice. (A and B) Representative images of mouse (A) and human (B) respiratory cilia stained for α-tubulin (a marker for the ciliary axoneme) and rabbit IgG (negative control, upper panel) or DNAH17 (lower panel). Scale bars represent 10 µm. (C) Quantitative real-time PCR analysis of Dnah17 mRNA expression in adult mouse tissues. Actb was used as an internal control. (D) Immunoblotting analysis of DNAH17 protein in different tissues from adult mice. GAPDH was used as the loading control. (E) Immunoblotting with sperm lysates from WT mice and epididymal lysates from Rpl10l +/− and Rpl10l −/− mice using the anti-DNAH17 antibody. Lamin B1 was used as the loading control. (F) Representative images of testicular tubules stained with anti-DNAH17 antibody and Hoechst showing that DNAH17 is localized in the cytoplasm and flagella of step 11–16 spermatids. Scale bars represent 50 µm. (G) Immunoblotting with lysates of human cell lines HepG2 (from liver), HEK293T (from embryonic kidney), HCT116 (from colon), A549 (from alveolar basal epithelia), U2OS (from bone), HeLa (from cervix), and adult human testes (hTestis) using the anti-DNAH17 antibody. GAPDH was used as the loading control. (H) Immunohistochemistry using the anti-DNAH17 antibody on adult human testicular sections with normal spermatogenesis. Rabbit IgG (left panel) was used as a negative control. Scale bars represent 50 µm. (I) Representative images of spermatozoa from fertile men (controls) stained with anti-DNAH17 antibody, anti–α-tubulin antibody, and Hoechst. Scale bar represents 10 µm. For A–I, three independent experiments were performed.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Expressing, Staining, Marker, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry

Localization of the mutant DNAH17 is not altered in patients. Representative images of spermatozoa from fertile controls and three patients stained with the anti-DNAH17 antibody, anti-α-tubulin antibody, and Hoechst. Two independent experiments were performed, and at least 150 sperm were examined for each time per individual. Scale bars represent 10 µm.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Localization of the mutant DNAH17 is not altered in patients. Representative images of spermatozoa from fertile controls and three patients stained with the anti-DNAH17 antibody, anti-α-tubulin antibody, and Hoechst. Two independent experiments were performed, and at least 150 sperm were examined for each time per individual. Scale bars represent 10 µm.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Mutagenesis, Staining

Generation of Dnah17 M/M mice modeling patients’ mutation and histological examinations of testes and epididymides from Dnah17 +/+ , Dnah17 +/M , and Dnah17 M/M mice. (A) Schematic illustrating construction of the mouse model ( Dnah17 M/M ). A gRNA was designed targeting exon 35 of Dnah17. The mutated nucleotides in mice (c.G5360A) and in patients (c.G5408A) are written in red. The nucleotide written in blue indicates a mutation not affecting the amino acid sequence in the protospacer adjacent motif, introduced by ssODNs. (B) Genomic DNA sequencing chromatograms showing the g.G39015A mutation heterozygous in Dnah17 +/M and homozygous in Dnah17 M/M mice. (C) cDNA sequencing chromatograms from Dnah17 +/M and Dnah17 M/M mice verified the c.G5360A mutation at mRNA level. (D) Periodic acid-Schiff staining of testicular sections showing that the spermatogenesis in Dnah17 M/M and Dnah17 +/M mice is comparable to that of Dnah17 +/+ mice. Scale bars represent 50 µm. (E) H&E staining of epididymal sections revealed similar sperm concentrations in Dnah17 M/M and Dnah17 +/M mice compared with that in Dnah17 +/+ mice. Scale bars represent 100 µm. (F) Histological examination of corpus epididymides from Dnah17 +/+ and Dnah17 M/M mice after ligation for 2 d and 4 d. Two independent experiments were performed. Scale bars represent 100 µm. (A–E) Three independent experiments were performed. Arrowheads, the mutation site; WT, the wild-type allele; MT, the mutant allele in humans; M, the mutant allele in mice.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Generation of Dnah17 M/M mice modeling patients’ mutation and histological examinations of testes and epididymides from Dnah17 +/+ , Dnah17 +/M , and Dnah17 M/M mice. (A) Schematic illustrating construction of the mouse model ( Dnah17 M/M ). A gRNA was designed targeting exon 35 of Dnah17. The mutated nucleotides in mice (c.G5360A) and in patients (c.G5408A) are written in red. The nucleotide written in blue indicates a mutation not affecting the amino acid sequence in the protospacer adjacent motif, introduced by ssODNs. (B) Genomic DNA sequencing chromatograms showing the g.G39015A mutation heterozygous in Dnah17 +/M and homozygous in Dnah17 M/M mice. (C) cDNA sequencing chromatograms from Dnah17 +/M and Dnah17 M/M mice verified the c.G5360A mutation at mRNA level. (D) Periodic acid-Schiff staining of testicular sections showing that the spermatogenesis in Dnah17 M/M and Dnah17 +/M mice is comparable to that of Dnah17 +/+ mice. Scale bars represent 50 µm. (E) H&E staining of epididymal sections revealed similar sperm concentrations in Dnah17 M/M and Dnah17 +/M mice compared with that in Dnah17 +/+ mice. Scale bars represent 100 µm. (F) Histological examination of corpus epididymides from Dnah17 +/+ and Dnah17 M/M mice after ligation for 2 d and 4 d. Two independent experiments were performed. Scale bars represent 100 µm. (A–E) Three independent experiments were performed. Arrowheads, the mutation site; WT, the wild-type allele; MT, the mutant allele in humans; M, the mutant allele in mice.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Mutagenesis, Sequencing, DNA Sequencing, Staining, Ligation

Characteristics of Dnah17 +/+ , Dnah17 +/M , and Dnah17 M/M male mice

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Characteristics of Dnah17 +/+ , Dnah17 +/M , and Dnah17 M/M male mice

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques:

Sperm flagella from Dnah17 M/M mice show frequent absence of MTDs 4–7 at principal piece and end piece. (A) Representative TEM micrographs showing cross sections of midpiece, principal piece, and end piece of sperm flagella from Dnah17 +/+ and Dnah17 M/M mice. (B) Percentages of the flagellar cross sections with loss of any combination of MTDs 4–7 at midpiece, principal piece, and end piece. (C and D) Frequencies of cross sections with MTD(s) 4, 5, 6, 7, 4+5, 4+7, 5+6, 5+7, 6+7, 4+5+6, 4+5+7, 4+6+7, 5+6+7, or 4+5+6+7 missing at principal piece (C) and end piece (D) from Dnah17 +/+ and Dnah17 M/M mice. (E) Representative cross sections with MTD 4 or 7 missing at principal piece and end piece of sperm flagella from Dnah17 M/M mice. (F) Percentages of flagellar cross sections with abnormalities other than MTD(s) 4–7 missing. For A and E, numbers in yellow indicate the MTDs with typical arrangement, numbers in red indicate the missing MTDs, and arrowheads highlight the ODAs; scale bars represent 200 nm. N, the number of mice examined. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. ***P < 0.001; Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: Sperm flagella from Dnah17 M/M mice show frequent absence of MTDs 4–7 at principal piece and end piece. (A) Representative TEM micrographs showing cross sections of midpiece, principal piece, and end piece of sperm flagella from Dnah17 +/+ and Dnah17 M/M mice. (B) Percentages of the flagellar cross sections with loss of any combination of MTDs 4–7 at midpiece, principal piece, and end piece. (C and D) Frequencies of cross sections with MTD(s) 4, 5, 6, 7, 4+5, 4+7, 5+6, 5+7, 6+7, 4+5+6, 4+5+7, 4+6+7, 5+6+7, or 4+5+6+7 missing at principal piece (C) and end piece (D) from Dnah17 +/+ and Dnah17 M/M mice. (E) Representative cross sections with MTD 4 or 7 missing at principal piece and end piece of sperm flagella from Dnah17 M/M mice. (F) Percentages of flagellar cross sections with abnormalities other than MTD(s) 4–7 missing. For A and E, numbers in yellow indicate the MTDs with typical arrangement, numbers in red indicate the missing MTDs, and arrowheads highlight the ODAs; scale bars represent 200 nm. N, the number of mice examined. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. ***P < 0.001; Student’s t test.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques:

MTDs 4–7 are destabilized in cauda epididymides of Dnah17 M/M mice. (A) Representative TEM micrographs of flagellar cross sections in testes from Dnah17 +/+ and Dnah17 M/M mice. (B) The percentages of cross sections with abnormal axoneme structure (including anomalies related to MTDs 4–7 and other anomalies) in testes from Dnah17 +/+ and Dnah17 M/M mice. (C – H) Representative TEM micrographs of flagellar cross sections and the percentages of cross sections with abnormal axoneme structure in caput (C and D), corpus (E and F), and cauda (G and H) epididymides from Dnah17 +/+ and Dnah17 M/M mice. Red arrowheads, cross sections with loss of any combination of MTDs 4–7; blue arrowheads, cross sections with axonemal abnormalities other than the loss of MTD(s) 4–7. Scale bars represent 500 nm. (I) The percentages of cross sections with loss of any combination of MTDs 4–7 in cauda epididymides of Dnah17 +/+ and Dnah17 M/M mice. (J) Immunoblotting with lysates of spermatozoa from cauda epididymides using anti-DNAH17, anti–α-tubulin, and anti–β-tubulin antibodies. Lamin B1 was used as the loading control. Three independent experiments were performed. N, the number of mice analyzed. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. ***P < 0.001; Student’s t test.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: MTDs 4–7 are destabilized in cauda epididymides of Dnah17 M/M mice. (A) Representative TEM micrographs of flagellar cross sections in testes from Dnah17 +/+ and Dnah17 M/M mice. (B) The percentages of cross sections with abnormal axoneme structure (including anomalies related to MTDs 4–7 and other anomalies) in testes from Dnah17 +/+ and Dnah17 M/M mice. (C – H) Representative TEM micrographs of flagellar cross sections and the percentages of cross sections with abnormal axoneme structure in caput (C and D), corpus (E and F), and cauda (G and H) epididymides from Dnah17 +/+ and Dnah17 M/M mice. Red arrowheads, cross sections with loss of any combination of MTDs 4–7; blue arrowheads, cross sections with axonemal abnormalities other than the loss of MTD(s) 4–7. Scale bars represent 500 nm. (I) The percentages of cross sections with loss of any combination of MTDs 4–7 in cauda epididymides of Dnah17 +/+ and Dnah17 M/M mice. (J) Immunoblotting with lysates of spermatozoa from cauda epididymides using anti-DNAH17, anti–α-tubulin, and anti–β-tubulin antibodies. Lamin B1 was used as the loading control. Three independent experiments were performed. N, the number of mice analyzed. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. ***P < 0.001; Student’s t test.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Western Blot

TEM analyses of sperm flagella in corpus epididymides after epididymal duct ligation. (A) Representative images of epididymides from Dnah17 +/+ and Dnah17 M/M mice after ligation for 2 d and 4 d. The epididymal ducts were ligated at the end of corpus adjacent to cauda. Each grid represents 1 mm. (B) Representative TEM micrographs of flagellar cross sections in corpus epididymides after ligation for 2 d and 4 d. Red arrowheads indicate cross sections with loss of any combination of MTDs 4–7, and blue arrowheads indicate cross sections with axonemal abnormalities other than the loss of MTD(s) 4–7. Scale bars represent 500 nm. (C) The percentages of cross sections with loss of any combination of MTDs 4–7 in corpus epididymides of Dnah17 +/+ and Dnah17 M/M mice. Two independent experiments were performed. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. **P < 0.01; one-way ANOVA test.

Journal: The Journal of Experimental Medicine

Article Title: A DNAH17 missense variant causes flagella destabilization and asthenozoospermia

doi: 10.1084/jem.20182365

Figure Lengend Snippet: TEM analyses of sperm flagella in corpus epididymides after epididymal duct ligation. (A) Representative images of epididymides from Dnah17 +/+ and Dnah17 M/M mice after ligation for 2 d and 4 d. The epididymal ducts were ligated at the end of corpus adjacent to cauda. Each grid represents 1 mm. (B) Representative TEM micrographs of flagellar cross sections in corpus epididymides after ligation for 2 d and 4 d. Red arrowheads indicate cross sections with loss of any combination of MTDs 4–7, and blue arrowheads indicate cross sections with axonemal abnormalities other than the loss of MTD(s) 4–7. Scale bars represent 500 nm. (C) The percentages of cross sections with loss of any combination of MTDs 4–7 in corpus epididymides of Dnah17 +/+ and Dnah17 M/M mice. Two independent experiments were performed. n, the number of axonemal cross sections analyzed. Data are presented as mean ± SEM. **P < 0.01; one-way ANOVA test.

Article Snippet: HEK293T cells (ATCC, CRL-3216) were transfected with pCR3 plasmids expressing 3xFlag-tagged human DNAH17 amino acids 3518–3817, mouse DNAH17 amino acids 3502–3801, human DNAH11 (amino acids 3572–3871), mouse DNAH11 (amino acids 3544–3843), human DNAH9 (amino acids 3542–3841), or mouse DNAH9 (amino acids 3540–3839), using lipofectamine 3000 (Invitrogen, L3000015).

Techniques: Ligation