7 research outputs found
Disrupted development of the sternum in <i>Spag17</i><sup><i>-/-</i></sup> mice.
<p>(A) Schematic representation of normal sternal bones (M, manubrium; S1 to S4, sternebrae 1 to 4; X, xiphoid). (B,C and D) Analysis of the sternum by micro-CT scanning shows fused sternebrae in the mutant mice. Pictures are representative images from respective supporting videos. (Fig 3B is representative of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125936#pone.0125936.s005" target="_blank">S1 Video</a>; Fig 3C is representative of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125936#pone.0125936.s006" target="_blank">S2 Video</a>; and Fig 3D is representative of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125936#pone.0125936.s007" target="_blank">S3 Video</a>.) (E and G) Alcian blue/ Alizarin red staining. Failure of ribs 4, 5 and 6 to complete their attachment to the sternum and promote sternebrae separation is apparent in the stained preparations in the <i>Spag17</i><sup><b><i>-/-</i></b></sup> mice (arrows). (F and H) Representative H&E staining shows the presence of normal cartilage development that prevents fusion of the vertebrae in wild type animals. (+/+), wild-type; (-/-), <i>Spag17</i>-mutant mice. * Fused sternebrae.</p
Primary cilia are altered in <i>Spag17</i>-deficient osteoblast, chondrocyte and MEFs cells.
<p>Cells were stained with anti-acetylated α-tubulin to visualize primary cilia and DAPI as a nuclei marker. (A) Primary cilia length from osteoblast cells. Scale bars, 5 μm. (B) Percentage of osteoblast cells expressing primary cilia. Scale bars, 20 μm (C) Primary cilia length from chondrocyte cells. Scale bars, 5 μm. (D) Percentage of chondrocyte cells expressing primary cilia. Scale bars, 20 μm. Mouse embryonic fibroblasts (MEFs) were isolated from embryos at E12.5. (E) Detection of <i>Spag17</i> mRNA and protein in MEFs. RT-PCR products were generated by primers from exon 4 and 5. The knockout mice have a deletion of the entire exon 5. Detection of SPAG17 protein by western blot using an antibody against C-terminal domain. (F) After serum-starvation to promote cilia growth, primary cilia were visualized in the MEFs with acetylated tubulin antibody, and DAPI as a nuclei marker. Primary cilia from <i>Spag17</i>-mutant MEFs were significantly shorter than wild-type. Scale bars, 5 μm. (G) <i>Spag17</i> knockdown in WT MEFs cells after treatment with <i>Spag17</i> siRNA duplex reproduced the shorter primary cilia phenotype. Scale bars, 5 μm. Arrows indicate primary cilia. (+/+), wild-type, (-/-); <i>Spag17</i>-mutant mice. Data are presented as means ± SEM. * Indicates statistically significant differences, p< 0.05.</p
Cranial and phalanges defects in <i>Spag17</i><sup><i>-/-</i></sup>.
<p>(A) Micro-CT imaging of mineralized skull. Note the demineralization of the skull in the mutant mouse (white arrows). (B and E) Quantitative mineralization measurement by micro-CT scanning. (C) Alkaline phosphatase activity in cultured calvarial osteoblast. (D) Micro-CT imaging of mineralized forelimb. Insert shows reduced metacarpal mineralization by Alcian Blue/ Alizarin Red staining. Micro-CT images were acquired in 360 projections by 360 degree rotation, with 680 millisecond exposure per projection. White and black arrows indicate less metacarpal mineralization in the mutant mouse. (+/+), wild-type; (-/-), <i>Spag17</i>-mutant mice. Data are presented as means ± SEM. * Indicates statistically significant differences, p< 0.05.</p
Tibia bone development is affected in <i>Spag17</i>-deficient mice.
<p>(A) Alcian Blue/ Alizarin Red staining on tibia from wild-type (+/+) and <i>Spag17</i>-mutant (-/-) mice. Measurement of tibia length and calcified tibia bone length shows <i>Spag17</i>-mutant limbs are shorter than wild-type mice. Scale bars, 0.5 mm. (B) Histological and morphometric studies on tibia shows cartilage and bone defects. Scale bars, 200 μm. (C) von Kossa staining. (D) Micro-CT structural analysis on tibias; mid-sagittal sections (left) and cross-sectional trans-axial views at the mid-diaphysis (right). Images were acquired in 600 projections in 180 degrees of rotation with 500 miliseconds exposure per projection. Scale bars, 200 μm. (E) mRNA expression of <i>osterix</i> (Osx) and <i>osteocalcin</i> (Ocn) measured by qPCR. (+/+), wild-type; (-/-), <i>Spag17</i>-mutant mice. Data are presented as means ± SEM from ≥7 mice. * Indicates statistically significant differences, p< 0.05. hz: hypertrophic zone; BA/TA: bone area/total area; MA/TA: marrow area/total area; BV/TV: bone volume/total volume; <i>Osx</i>: <i>osterix</i>; <i>Ocn</i>: <i>osteocalcin</i>.</p
Video1_SPAG17 mediates nuclear translocation of protamines during spermiogenesis.MP4
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.</p
DataSheet1_SPAG17 mediates nuclear translocation of protamines during spermiogenesis.pdf
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.</p
Video2_SPAG17 mediates nuclear translocation of protamines during spermiogenesis.MP4
Protamines (PRM1 and PRM2) are small, arginine-rich, nuclear proteins that replace histones in the final stages of spermiogenesis, ensuring chromatin compaction and nuclear remodeling. Defects in protamination lead to increased DNA fragmentation and reduced male fertility. Since efficient sperm production requires the translocation of protamines from the cytoplasm to the nucleus, we investigated whether SPAG17, a protein crucial for intracellular protein trafficking during spermiogenesis, participates in protamine transport. Initially, we assessed the protein-protein interaction between SPAG17 and protamines using proximity ligation assays, revealing a significant interaction originating in the cytoplasm and persisting within the nucleus. Subsequently, immunoprecipitation and mass spectrometry (IP/MS) assays validated this initial observation. Sperm and spermatids from Spag17 knockout mice exhibited abnormal protamination, as revealed by chromomycin A3 staining, suggesting defects in protamine content. However, no differences were observed in the expression of Prm1 and Prm2 mRNA or in protein levels between testes of wild-type and Spag17 knockout mice. Conversely, immunofluorescence studies conducted on isolated mouse spermatids unveiled reduced nuclear/cytoplasm ratios of protamines in Spag17 knockout spermatids compared to wild-type controls, implying transport defects of protamines into the spermatid nucleus. In alignment with these findings, in vitro experiments involving somatic cells, including mouse embryonic fibroblasts, exhibited compromised nuclear translocation of PRM1 and PRM2 in the absence of SPAG17. Collectively, our results present compelling evidence that SPAG17 facilitates the transport of protamines from the cytoplasm to the nucleus.</p