Journal: iScience

Article Title: Loss of histone reader Phf7 leads to immune pathways activation via endogenous retroviruses during spermiogenesis

doi: 10.1016/j.isci.2023.108030

Figure Lengend Snippet:

Article Snippet: Rabbit Lamin B1 Polyclonal , bioss , bs-1840R.

Techniques: ROS Assay, TUNEL Assay, Apoptosis Assay, Bicinchoninic Acid Protein Assay, Recombinant, SYBR Green Assay, Gene Knockout, Software

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Quantitative real-time PCR primer sequences

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is expressed in human hepatocytes during early gestation but not in normal perinatal or adult hepatocytes. A–C: immunohistochemical staining of GATA6 protein in normal human liver. Brown indicates positive staining in nuclei. In fetal liver (FL) at gestational week (GW) 13 (FL GW13), GATA6 protein is expressed in all hepatocytes (arrowhead) and other cell types of the liver (A). At GW37, GATA6 protein expression is diminished in hepatocytes (arrowhead) and restricted to cholangiocytes lining the bile ducts (arrow) (B). The expression pattern of GATA6 protein in adult liver (AL) is similar to that of late FL with positive cholangiocytes (arrow) and negative hepatocytes (arrowhead) (C). D–G: in situ hybridization of GATA6 mRNA in FL GW37 (D and E) and AL (F and G). Green indicates positive signal in cytoplasm. Cholangiocytes show positive signal (arrow); hepatocytes show weak or negative signal (arrowhead). Scale bar = 200 µm (A–C) and 20 µm (D–G).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Immunohistochemical staining, Staining, Expressing, In Situ Hybridization

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Hepatocyte expression of GATA6 is elevated in biliary atresia (BA) and decreased after portoenterostomy (PE). A: boxplot showing the relative GATA6 mRNA expression, as determined by qPCR, in different liver sample groups. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01 compared with BA group. The P values of other paired comparisons are indicated in Table 2. B–E: in situ hybridization of GATA6 in BA samples demonstrates strongly positive hepatocytes (arrowhead) (B and C), whereas in situ hybridization in BA-post-PE samples shows weak or negligible signal in hepatocytes (arrowhead) (D and E). F–H: GATA6 immunohistochemistry from normal adult liver (AL) with negative hepatocytes and positive cholangiocytes (F), BA with strong immunoreactivity in hepatocyte nuclei (G), and BA-post-PE with less immunoreactivity in hepatocytes compared with BA (H). I: Western blotting of GATA6 protein in AL, BA, BA-post-PE, and disease control (DC) samples. NBI, normalized band intensity. J: paired sample analysis of GATA6 protein expression from patients before BA and after PE (BA-post-PE). Green (in situ hybridization) and brown (immunohistochemistry) indicate positive staining. Scale bars = 150 µm (B and C), 20 µm (C and E), and 200 µm (F and H).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Western Blot, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: P values from comparisons of mRNA levels of different liver sample types (in Figs. 2A and 7A, C, and E) using Wilcoxon method

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 immunoreactivity in liver sample groups

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Double in situ hybridization in biliary atresia (BA) liver. A and B: double in situ hybridization was performed on 2 different samples of BA liver. GATA6 expression is high in both ductular reaction (DR) area and liver parenchyma (hepatocytes); these 2 histological compartments are denoted in the figures by dashed lines. C and D: CK7 (C) is strongly expressed in DR area, whereas its expression is weaker in hepatocytes. CFTR (D) expression is limited to DR area. E and F: merged images of DAPI, GATA6, and CK7 (E) or DAPI, GATA6, and CFTR (F). Bile duct epithelium (arrow) is positive for both GATA6 and CK7/CFTR, but hepatocytes (arrowhead) express only GATA6. G and I: higher-magnification images from E. H and J: higher-magnification images from F. Green indicates positive signal for GATA6, and red indicates positive signal for CK7 or CFTR. Blue indicates DAPI staining in the nuclei of all cell types. Scale bars = 50 µm (A–F) and 10 µm (G–J).

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: In Situ Hybridization, Expressing, Staining

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: GATA6 is overexpressed in hepatocytes in 2 mouse models of biliary obstruction. A: GATA6 protein is expressed in bile duct epithelium (arrow) but not in hepatocytes (arrowhead) of normal murine liver (4 wk after sham surgery). B: in mice subjected to bile duct ligation (BDL), hepatocytes (arrowhead) in the periportal area strongly express GATA6. C and D: at postnatal day 15 (P15), before the onset of ductular reaction (DR), there remains weak expression of GATA6 in normal hepatocytes (arrowhead), and there is no difference in GATA6 expression between control (Ctrl) and Alb-Cre;Rbpjflox/flox;Hnf6flox/flox double knockout (DKO) mice. E and F: at P30 Ctrl hepatocytes are GATA6 negative, whereas the Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver with severe DR shows strong GATA6 immunoreactivity in hepatocytes. G and H: at P120 in Alb-Cre;Rbpjflox/flox;Hnf6flox/flox liver, the DR has diminished, and liver histology, as well as GATA6 expression, is similar to Ctrl liver with immunoreactivity only in bile duct epithelial cells (arrow). n = 3 in each group. Scale bars = 50 µm.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Ligation, Expressing, Double Knockout

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: In patients with biliary atresia (BA), GATA6 protein expression in hepatocytes correlates to bile duct expansion (BDE), age at portoenterostomy (PE), and the liver injury marker alanine aminotransferase. Hepatocyte GATA6 protein expression was divided into 2 groups (<70% positive nuclei = low/intermediate vs. >70% positive nuclei = high) and correlated to BDE rate (P = 0.0094) (A), age at PE (B), and plasma (P)-alanine aminotransferase (C). A: contingency tabling coupled with χ2 test was employed to test the statistical significance. B and C: dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Marker

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Enforced expression of GATA6 causes alterations in expression of genes regulating cholangiocyte and hepatocyte differentiation. The human hepatocellular cell line HepG2 and primary human hepatocytes were transiently transfected with pCDNA3-GATA6 or pCDNA3 plasmid alone. After 48 h, RNA was harvested and subjected to qRT-PCR analysis. A: in HepG2 cells, 3 genes related to cholangiocyte differentiation (HNF1β, HNF6, and JAG1) were significantly upregulated in cells overexpressing GATA6 compared with control (Ctrl) cells. B: in primary human hepatocytes, the relative expression of 4 genes related to cholangiocyte differentiation (HNF1β, HNF6, JAG1, and DKK1) and 1 gene related to hepatocyte differentiation (HNF4α) were upregulated in cells with GATA6 overexpression compared with control cells. For both cell types, 5 independent experiments were performed in triplicate. Each bar depicts the logarithm of the ratio of mRNA expression GATA6 overexpression vs. control cells. *P < 0.05, **P < 0.01.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Over Expression

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Transcription factor GATA6: a novel marker and putative inducer of ductal metaplasia in biliary atresia

doi: 10.1152/ajpgi.00362.2017

Figure Lengend Snippet: Expression of HNF1β, HNF6, and JAG1 is elevated in biliary atresia (BA) liver, decreases after successful portoenterostomy (PE), and correlates to GATA6. A, C, and E: boxplots showing relative mRNA expression of HNF1β (A), HNF6 (C), and JAG1 (E), as measured by qPCR, in BA vs. other liver specimens. Dots represent individual samples, the box represents the interquartile range, and the whiskers represent the 1st and 4th quartile. The line inside the box is the median, and the dashed line represents the mean. *P < 0.05, **P < 0.01. The P values of other paired comparisons are indicated in Table 2. B, D, and F: linear regression analyses of HNF1β (B), HNF6 (D), and JAG1 (F) mRNA expression vs. GATA6 mRNA expression in BA-post-PE samples. FL, fetal liver; AL, adult liver; DC, disease control.

Article Snippet: Primary antibodies used were as follows: rabbit antihuman GATA6 IgG (sc-9055; Santa Cruz Biotechnologies, Santa Cruz, CA) at dilution 1:1,200 for human samples, goat anti-human GATA6 IgG (AF1700; R&D Systems, Minneapolis, MN) at dilution 1:1,200 for mouse samples.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Investigating Dcaf17 mRNA levels by real time PCR. ( A ) Relative expression of Dcaf17 in different tissues of adult mouse. Relative expression values of Dcaf17 mRNA were normalized to 18S rRNA expression and the level of Dcaf17 mRNA in the brain was arbitrarily set at 1. The Dcaf17 is highly expressed in testis, while other tissues show low level of expression. ( B ) Relative expression of Dcaf17 in testis at different ages. Relative expression values of Dcaf17 mRNA were normalized to 18S rRNA expression and the level of Dcaf17 mRNA in the 5 days postpartum (dpp) testis was arbitrarily set at 1. Expression of Dcaf17 in the testis increased with the age till the age of 32 dpp and then after it remained constant. Asterisks indicate statistical significance (p < 0.0001 for A and p < 0.0036 for B). Error bars represent SEM. Three to five animals were used to investigate the transcript levels of Dcaf17 . Experiments were performed in triplicates. Data were analyzed on Graphpad prism 5 software using one-way ANOVA technique followed by post hoc Bonferroni’s ( A ) or Tukey’s ( B ) multiple comparison test. ns - not significant.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Software, Comparison

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Diagrammatic representation (A) of Dcaf17 gene targeting approach in mouse by homologous recombination and genotyping ( B , C ) of different alleles of Dcaf17 in mice. ( A ) Homologous recombination strategy in mouse ES cells. The Dcaf17 targeting vector (top) was constructed to replace wild type exon 4 and introduce neomycin drug selection marker, LoxP and FRT sites. ( B ) Agarose gel image of PCR genotyping of representative Dcaf17 mutant mice. PCR amplification of wild type genotype gives 1 kbps amplicon (1, C3), heterozygous genotype for Dcaf17 mutation gives 1 kbps and 193 bps amplicons (3–5, C1) and homozygous genotype for Dcaf17 mutation gives 193 bps amplicon (2, 6 and C2). 1–6 – genomic DNA samples of different Dcaf17 genotypes; C1-C3 – Different Dcaf17 genotype controls; −Ve – no template control. ( C ) Agarose gel image of RT-PCR of different Dcaf17 genotypes. PCR products of various Dcaf17 alleles and β-actin were run on the same agarose gel and single image was taken. +/+ - Dcaf17 +/+ (WT); +/− - Dcaf17 +/− (heterozygous Dcaf17 mutant); −/− - Dcaf17 −/− (homozygous Dcaf17 mutant); −Ve – no template control; M – DNA ladder. PCR fragment size for β-actin is 190 bps; for Dcaf17 +/+ is 284 bps and for Dcaf17 −/− is 148 bps. Gel images were taken using ImageQuant LAS 4000 imaging system.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Homologous Recombination, Plasmid Preparation, Construct, Introduce, Selection, Marker, Agarose Gel Electrophoresis, Mutagenesis, Amplification, Control, Reverse Transcription Polymerase Chain Reaction, Imaging

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Fertility testing of Dcaf17 −/− and wild type mice.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques:

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Histology of cauda epididymides and cauda sperm analyses from WT and Dcaf17 −/− adult mice. Hematoxylin and eosin ( H , E ) staining of cauda epididymides sections (5 µm thick) from 8 weeks ( A , B ) and 8 months ( C , D ) old WT ( A , C ) and Dcaf17 −/− ( B , D ) mice. The lumen of WT cauda epididymis ( A , C ) is full of mature sperm, whereas the lumen of Dcaf17 −/− mutant cauda epididymidis ( B , D ) contains oval shaped sloughed cells of various size. Sperm count ( E ) and motility ( F ) analyses of WT and Dcaf17 −/− mutant mice at the age of 8 weeks and 8 months old reveal severe and progressive reduction in sperm count ( E ) and motility ( F ) in Dcaf17 −/− mice compared to WT mice. Around 300 sperm in a total of five fields in each replicate were analyze for sperm motility analysis. Number of animals used for each genotype to analyze sperm count ( E ) and motility ( F ) was 5. Data were analyzed on Graphpad prism 5 using two-way ANOVA technique followed by post hoc Bonferroni’s multiple comparison test. For images A to D the magnification is 200X and the scale bars are 5 µm. Error bars in panels E and F represent SEM. Asterisks in E and F indicate statistical significance (p < 0.0001 for E and F).

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Staining, Mutagenesis, Comparison

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Bright field and fluorescence microscopy of cauda epididymal sperm from WT and Dcaf17 −/− adult mice. Representative images of Diff-Quick staining (bright field images, top panels) and MitoTracker staining (fluorescence images, bottom panels) of epididymal sperm spreads from WT ( A , C ) and Dcaf17 −/− ( B , D ) adult mice. The WT sperm ( A , C ) show typical hook-shaped head, patent midpiece and tail morphology. Whereas, the Dcaf17 −/− sperm ( B , D ) show variety of morphological defects in head shape, midpiece and tail. To categorize different sperm defects in Dcaf17 KO mice we analyzed total 150 sperms from 3 different mice. Major categories of sperm head defects were triangular (cupcake) ( B1 , D1 , D3 ), oval ( B5 , D2 , D4 , D5 ) or amorphous ( B2 , B3 ) shaped with many times either bent head or coiled midpiece surrounding the head. Fluorescently labeled WT sperm ( C ) shows normal crescent shaped acrosome (green), uniformly distributed mitochondria (red) along the midpiece and highly condensed nucleus (blue). Dcaf17 −/− sperm ( D1–5 ) show spatially dysmorphic sperm structure with abnormal acrosome (green), ectopic localization of mitochondria (red) and diffused chromatin structure (blue). Images C and D1–5 are merged fluorescence images of sperm stained for acrosome (green), midpiece (red) and nucleus (blue). Separate fluorescence images for each sperm acrosome, midpiece and nucleus staining are shown in supplementary figure . Magnification of bright images ( A , B1–5 ) is 400X and magnification of fluorescence images ( C , D1–5 ) is 1000X. Scale bar in the image A is 1 mm and in the image C is 10 µm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Fluorescence, Microscopy, Diff-Quik, Staining, Labeling

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Transmission electron microscopy (TEM) of cauda epididymides from 8 weeks old WT ( A–C ) and Dcaf17 −/− ( D–I ) adult mice. Representative TEM images of WT cauda epididymis ( A–C ) show spermatozoa with typical elongated nucleus (Nu) with homogeneously condensed chromatin. The acrosome (Ac) in WT sperm is covering the anterior portion of the head and is tightly attached to the nucleus through the acrosome-acroplaxome complex. The mid-piece of WT sperm show spirally arranged mitochondria (Mt) that are enclosed by well-defined mitochondrial sheath. WT sperm tail sections show the typical “9 + 2” pattern of the microtubular axoneme (MA). TEM images of Dcaf17 −/− cauda epididymis ( D–I ) show abnormal sperm with misshaped head containing defective nuclear (Nu) chromatin condensation, malformed and detached acrosome (Ac), disorganized mitochondria (Mt) trapped inside large cytoplasmic droplets and ectopic localization of the microtubular axoneme (MA). Inset image in panel B is enlarged image of squared region of image B. Image E is a higher magnification image of square region shown in the image D. Scale bar are shown at the bottom right corner to each image. Scale bar: A, D 5 µm; B, C, E, F, and G 2 µm; H and I 1 µm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Transmission Assay, Electron Microscopy

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Histology of testes and cauda epididymides from WT and Dcaf17 −/− mice at different ages. Sections (5 µm thick) of testes and cauda epididymes from WT and Dcaf17 −/− mice at different ages were stained with Hematoxylin and Eosin. Testes of WT ( A ) and Dcaf17 −/− ( B ) mice at 5 days postpartum (dpp) show normal Sertoli cells with no apparent morphological or histological differences. At 14 dpp, normal spermatocytes are seen in the WT ( C ) and Dcaf17 −/− ( D ) testes sections. Unlike WT testis at 14 dpp, the lumen of Dcaf17 −/− seminiferous tubules (ST) ( D ) show large shaded cells (arrow heads). Spermatocytes at most advanced stage and round spermatids are observed in the ST of the WT ( E ) and Dcaf17 −/− ( F ) testes at 23 dpp. The lumen of ST in Dcaf17 −/− testis at 23 dpp ( F ) shows giant, multinucleated and prematurely sloughed cells (arrow heads). Epithelial vacuoles (arrow) are also seen in the Dcaf17 −/− testis at 23 dpp. At 32 dpp, the WT testis ( G ) shows numerous elongating and elongated spermatids with normal morphology and chromatin condensation. Dcaf17 −/− testis at 32 dpp ( H ) shows fewer elongating and elongated spermatids with abnormal shape and chromatin condensation. Testes from 42 dpp ( I ) and 56 dpp ( K ) old WT mice show a complete cycle of spermatogenesis with seminiferous tubules depicting various stages of highly organized spermatogenic cells. Testes of Dcaf17 −/− mice at 42 dpp ( J ) and 56 dpp ( L ) show abnormal spermatogenesis with sever defects in post-meiotic stages of spermatogenesis. Vacuoles (arrows) are observed in the epithelium of some ST of the Dcaf17 −/− testis ( J , L ). The lumen of cauda epididymis ( M ) from sexually immature (14 dpp) WT mouse is devoid of any degenerated cells. In Dcaf17 −/− mouse at 14 dpp, the lumen of cauda epididymis shows numerous prematurely degenerated oval shaped cells of various size. Sexually mature cauda epididymis of WT mouse (32 dpp) has lumen ( O ) full of normal mature sperm. Prematurely degenerated spermatogenic cells of various size and few abnormal sperm are observed in the lumen of Dcaf17 −/− mouse cauda epididymis ( P ) at 32 dpp. Magnification 400X. Scale bar 2 mm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Staining

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Periodic acid-Schiff (PAS) staining of testes sections from 8 weeks old WT and Dcaf17 −/− mice. To visualize the glycoproteins/acrosomes (violet) and nuclei (blue), the testis sections from WT ( A–C ) and Dcaf17 −/− ( D–F ) mice were stained with PAS-stain and hematoxylin counter stain. WT testis sections ( A–C ) show normal spermatogenesis with well-organized stages of germ cell development, round spermatids with PAS-positive normal acrosomal caps (arrows), elongating and elongated spermatids (arrow heads) and chromatin condensation. Dcaf17 −/− testis sections ( D–F ) show defective spermatogenesis with abnormal acrosomal caps (arrows), distorted elongated spermatids (arrows heads) and chromatin condensation. Magnification – 1000X. Scale bar: 10 µm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Staining

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: TUNEL staining of testes sections from 8 weeks old WT and Dcaf17 −/− mice to assess germ cell apoptosis. Testes sections of 8 weeks old WT ( A–C ) and Dcaf17 −/− ( D–F ) mice were subjected to TUNEL assay, which detects fragmented DNA in the apoptotic germ cells (green). WT testis section ( A–C ) shows fewer TUNEL-positive cells (green) ( B , C ) compare to Dcaf17 −/− testis section ( E , F ). Image H is positive control for TUNEL assay where the testis section was treated with DNaseI enzyme to generate fragmented genomic DNA. Image K is a negative control where testis section was treated only with labelling solution without terminal transferase. Images A,D,G and J are bright field images of respective TUNEL stained fluorescence images B, E, H, and K. Images C,F,I,L are merged images of respective bright field and fluorescence images. Magnification 200X. Scale Bar ( J ): 50 µm. The number of TUNEL-positive cells (green) per tubule in the testes sections of WT and Dcaf17 −/− mice aged 8 weeks and 8 months were plotted in the graph ( M ). The Histogram ( M ) represents mean ± SEM of 3 animals analyzed for TUNEL assay from each strain. Asterisks indicate statistical significance (p < 0.001 at 8 weeks and P < 0.01 at 8 month). Data were analyzed on Graphpad prism 5 using two-way ANOVA technique followed by post hoc Bonferroni’s multiple comparison test.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: TUNEL Assay, Staining, Positive Control, Negative Control, Fluorescence, Comparison

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Immunofluorescence analysis of meiotic spermatocytes of 8 weeks old WT and Dcaf17 −/− testes. Immunostaining of the synaptonemal complex with SYCP1 (green) and SYCP3 (red) antibodies in pachytene spermatocytes of WT and Dcaf17 mutant mice (Panel A). Immunofluorescnece staining of γ-H2AX (red) and the lateral element of the synaptonemal complex, SYCP3 (Green) in pachytene spermatocytes of WT and Dcaf17 −/− mice (Panel B). Spermatocytes with synaptic X and Y chromosomes (green) around which γ-H2AX (red) was normally distributed in WT and Dcaf17 −/− strains. Nucleus is counter stained with DAPI (blue). Magnification 1000X. Scale bar: 10 µm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Immunofluorescence, Immunostaining, Mutagenesis, Staining

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Manchette and acrosome formation based on α-tubulin and PNA immunofluorescence staining of testes squash preparations from WT and Dcaf17 −/− mice. Testes squash preparations from WT and Dcaf17 −/− testes from adult mice were stained using antibodies against α-tubulin (red) and lectin PNA (green) to analyze manchette and acrosome formation, respectively. Nucleus was stained with DAPI (blue). Representative fluorescence images of microtubule (red), acrosome (green) and nucleus (blue) staining and corresponding merge images of post-meiotic male germ cells at different steps of spermiogenesis are shown for both the genotypes (WT and Dcaf17 −/− ) at similar stages. Different steps of sperimioenesis are depicted on the basis of nuclear and acrosome staining. WT post-meiotic germ cells show normal microtubule bundles (manchette) assembly (red), acrosome morphogenesis (green) and nuclear (blue) elongation during spermiogenesis. In the Dcaf17 mutant spermiogenic cells, the organization of microtubule bundles (red) around the nucleus (blue) is disrupted resulting in abnormal manchette formation, defective nuclear (blue) condensation and elongation, and abnormal acrosome (green) morphogenesis. Ectopic microtubules, defective acrosome and abnormal nuclear head shaping are noticeable in Dcaf17 −/− post-meiotic germ cells. Magnification 1000X. Scale bar: 10 µm. Schematic representations of microtubule bundles (red) assembly, acrosome (green) formation in relation to the nucleus (blue) shaping during spermiogenesis in wild-type and Dcaf17 KO spermatids are shown.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Mutagenesis

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: Testicular ultrastructure of WT and Dcaf17 −/− adult mice by transmission electron microscopy (TEM). Representative TEM images of 8 weeks old WT ( A–F ) and Dcaf17 −/− ( G–R ) mice testes ultrathin sections displaying different steps of spermiogenesis. Spermatids in step 3–5 ( A , G , M ), step 6–7 ( B , H , N ), step 8–9 ( C , I , O ), step 10 ( D,J,P ), step 11–12 ( E , K , Q ) and step 15–16 ( F , L , R ) are represented. In WT ( A ) and Dcaf17 −/− ( G , M ) testes, round spermatids during steps 3–5( A , G , M ) show acrosome (Ac) attached with spherical nucleus (Nu) at one end. Some round spermatids show Golgi (Go) apparatus located close to acrosome (Ac). During steps 6 and 7 of spermiogenesis in WT ( B ) and Dcaf17 −/− ( H , N ) testes, the acrosome (Ac) flattens and grows to form a cap covering approximately half of the nuclear (Nu) surface. During steps 8–12 of WT spermiogenesis, the spermatid nucleus (Nu) starts to elongate and the acrosome (Ac) extends along the nuclear envelope ( C , D , E ). The manchette (Mn) in WT elongating spermatids ( C , D , E ) extends from perinuclear ring (PR) into residual body (RB) and attached to posterior region of nuclear membrane. The Dcaf17 −/− elongating spermatids in steps 8–12 ( I–K and O–Q ) show abnormal nuclear elongation and asymmetric extension of the acrosome (Ac) that is associated with distorted nucleus (Nu). The perinuclear ring (PR) in the elongated spermatids of Dcaf17 −/− mutant ( I–K and O–Q ) is abnormally positioned and microtubule bundles of manchette (Mn) are severely disorganized along the posterior surface of the deformed nucleus (Nu). WT elongated spermatids in steps 15–16 show elongated nucleus (Nu), a midpiece (MP) region containing uniformly distributed mitochondria (Mt) and connecting piece (CP) joining the midpiece region of the tail to the head of maturing spermatozoa. The manchette (Mn) and residual body (RB) gradually disappear during the maturation steps 15–16 in WT. Elongated spermatids at steps 15–16 in Dcaf17 −/− testis (L,R) show oval shaped nucleus (Nu), abnormal manchette (Mn) ( L ) and residual body (RB). Scale bars: ( A–E) , ( G–K) and ( M–R) 5 µm; F and L 2 µm.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Transmission Assay, Electron Microscopy, Membrane, Mutagenesis

Journal: Scientific Reports

Article Title: Deletion of DDB1- and CUL4- associated factor-17 ( Dcaf17) gene causes spermatogenesis defects and male infertility in mice

doi: 10.1038/s41598-018-27379-0

Figure Lengend Snippet: List of primers used in this study.

Article Snippet: Conditional Dcaf17 knockout (KO) mice were generated by inGenious Targeting Laboratory, Inc. (Ronkonkoma, NY, USA).

Techniques: Sequencing, Plasmid Preparation