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Sadaki Yokota - One of the best experts on this subject based on the ideXlab platform.
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Argonaute2 Protein in Rat Spermatogenic Cells Is Localized to Nuage Structures and LAMP2-Positive Vesicles Surrounding Chromatoid Bodies.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2016Co-Authors: Yuki Fujii, Hideaki Fujita, Yuko Onohara, Sadaki YokotaAbstract:Localization of Argonaute2 (AGO2) protein--an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in Nuage of rat spermatogenic cells--was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of Nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of Nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas Nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments.
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ddx6 localizes to Nuage structures and the annulus of mammalian spermatogenic cells
Histochemistry and Cell Biology, 2014Co-Authors: Chika Kawahara, Sadaki Yokota, Hideaki FujitaAbstract:The localization of DEAD (Asp-Glu-Ala-Asp) box helicase 6 (DDX6) in spermatogenic cells from the mouse, rat, and guinea pig was studied by immunofluorescence (IF) and immunoelectron microscopy (IEM). Spermatogenic cells from these species yielded similar DDX6 localization pattern. IF microscopy results showed that DDX6 localizes to both the nucleus and cytoplasm. In the cytoplasm of spermatogenic cells, diffuse cytosolic and discrete granular staining was observed, with the staining pattern changing during cell differentiation. IEM revealed that DDX6 localized to the five different types of Nuage structures and non-Nuage structures, including small granule aggregate and late spermatid annuli. Nuclear labeling was strongest in leptotene and zygotene spermatocytes and moderately strong in the nuclear pocket of late spermatids. DDX6 also localized to the surface of outer dense fibers, which comprise of flagella. The results show that DDX6 is present in Nuage and non-Nuage structures as well as nuclei, suggesting that DDX6 has diverse functions in spermatogenic cells.
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expression of mael in Nuage and non Nuage compartments of rat spermatogenic cells and colocalization with ddx4 ddx25 and miwi
Histochemistry and Cell Biology, 2013Co-Authors: Miki Takebe, Yuko Onohara, Sadaki YokotaAbstract:The functions of MAELSTROM protein (MAEL) in spermatogenesis are gradually being identified but the precise distribution of MAEL in spermatogenic cells during spermatogenesis has not yet been mapped. We studied the expression of MAEL in rat testis by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence staining showed that MAEL was localized in intermitochondrial cement, irregularly-shaped perinuclear granules and satellite bodies of pachytene spermatocytes, and in chromatoid bodies of spermatids. The SBs appeared exclusively in pachytene spermatocytes at stages IX-X and were stained strongly for MAEL. In step 12-19 spermatids, many granules stained for MAEL but not DDX4. These granules were confirmed to be non-Nuage structures, including mitochondria-associated granules, reticulated body, granulated body by IEM. In the neck region of late spermatids and sperm, MAEL-positive small granules were found. MAEL is colocalized with MIWI in Nuage and non-Nuage. The results suggest that MAEL seems to function in Nuage and non-Nuage structures and interacts with MIWI.
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Nuage proteins: their localization in subcellular structures of spermatogenic cells as revealed by immunoelectron microscopy
Histochemistry and Cell Biology, 2012Co-Authors: Sadaki YokotaAbstract:Chromatoid body (CB) was identified as granules stained by basic dye 130 years ago and called by various names. Electron microscopy revealed that the CB belonged to Nuage (cloud in French) specific for germ cells. We described the localization of several proteins, including RNA helicases, in the Nuage compartments classified into six types and in several spermatogenic cell-specific structures. All the proteins examined were detected in the Nuage, including the CB with different staining intensities. Several proteins were localized to non-Nuage structures, suggesting that these Nuage proteins structures are related to Nuage function.
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Nuage components and their contents in mammalian spermatogenic cells as revealed by immunoelectron microscopy
2012Co-Authors: Yuko Onohara, Sadaki YokotaAbstract:The Nuage is a germ cell-specific organelle that has been studied for more than a hundred years. It was first discovered in spermatogenic cells as a perinuclear granule stained by basic dyes and visualized using a light microscope. Morphological studies using electron microscopes demonstrated that ”chromatoid body (CB)” is a Nuage component in spermatids, and that “inter-mitochondrial cement (IMC)” is another Nuage component found in spermatocytes. During meiosis, the IMC disappears, but the CB soon reforms in post-meiotic spermatids. Recent morphological and molecular biological studies have identified many components of Nuage, and suggest that they play roles in the silencing, decay, and storage of RNA, and in the aggresome system. In this report, we summarize recent findings related to Nuage and discuss their functions in spermatogenic cells.
Toshie Kai - One of the best experts on this subject based on the ideXlab platform.
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the tudor domain protein kumo is required to assemble the Nuage and to generate germline pirnas in drosophila
The EMBO Journal, 2012Co-Authors: Amit Anand, Toshie KaiAbstract:In Drosophila ovaries, distinct Piwi-interacting RNA (piRNA) pathways defend against transposons in somatic and germline cells. Germline piRNAs predominantly arise from bidirectional clusters and are amplified by the ping-pong cycle. In this study, we characterize a novel Drosophila gene, kumo and show that it encodes a conserved germline piRNA pathway component. Kumo contains five tudor domains and localizes to Nuage, a unique structure present in animal germline cells, which is considered to be the processing site for germline piRNAs. Transposons targeted by the germline piRNA pathway are derepressed in kumo mutant females. Moreover, germline piRNA production is significantly reduced in mutant ovaries, thereby indicating that kumo is required to generate germline piRNAs. Kumo localizes to the Nuage as well as to nucleus early female germ cells, where it is required to maintain cluster transcript levels. Our data suggest that kumo facilitates germline piRNA production by promoting piRNA cluster transcription in the nucleus and piRNA processing at the Nuage.
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pirna pathway and the potential processing site the Nuage in the drosophila germline
Development Growth & Differentiation, 2012Co-Authors: Jun Wei Pek, Veena S Patil, Toshie KaiAbstract:The accurate transfer of genetic material in germline cells during the formation of gametes is important for the continuity of the species. However, animal germline cells face challenges from transposons, which seek to spread themselves in the genome. This review focuses on studies in Drosophila melanogaster on how the genome protects itself from such a mutational burden via a class of gonad-specific small interfering RNAs, known as piRNAs (Piwi-interacting RNAs). In addition to silencing transposons, piRNAs also regulate other processes, such as chromosome segregation, mRNA degradation and germline differentiation. Recent studies revealed two modes of piRNA processing – primary processing and secondary processing (also known as ping-pong amplification). The primary processing pathway functions in both germline and somatic cells in the Drosophila ovaries by processing precursor piRNAs into 23–29 nt piRNAs. In contrast, the secondary processing pathway functions only in the germline cells where piRNAs are amplified in a feed-forward loop and require the Piwi-family proteins Aubergine and Argonaute3. Aubergine and Argonaute3 localize to a unique structure found in animal germline cells, the Nuage, which has been proposed to function as a compartmentalized site for the ping-pong cycle. The Nuage and the localized proteins are well-conserved, implying the importance of the piRNA amplification loop in animal germline cells. Nuage components include various types of proteins that are known to interact both physically and genetically, and therefore appear to be assembled in a sequential order to exert their function, resulting in a macromolecular RNA-protein complex dedicated to the silencing of transposons.
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drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microrna 7
Developmental Cell, 2009Co-Authors: Jun Wei Pek, Ai Khim Lim, Toshie KaiAbstract:Nuage is a germline-unique perinuclear structure conserved throughout the animal kingdom. Maelstrom (Mael) is an unusual Nuage component, as it is also found in the nucleus. Mael contains a High Mobility Group box, known to mediate DNA binding. We show that Mael nuclear function is required for proper differentiation in the Drosophila germline stem cell (GSC) lineage. In mael mutant testes, transit-amplifying cysts fail to differentiate into primary spermatocytes, instead breaking down into ectopic GSCs and smaller cysts, due to a depletion of Bag-of-marbles (Bam) protein. Mael regulates Bam via repression of miR-7. Mael binds the miR-7 promoter and is required for the local accumulation of HP1 and H3K9me3. miR-7 targets bam directly at its 3'UTR, and a reduction in miR-7 expression can rescue germline differentiation defects found in mael mutants by alleviating Bam repression. We propose that Mael ensures proper differentiation in the GSC lineage by repressing miR-7.
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unique germ line organelle Nuage functions to repress selfish genetic elements in drosophila melanogaster
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Ai Khim Lim, Toshie KaiAbstract:The Nuage is an electron-dense perinuclear structure that is known to be a hallmark of animal germ-line cells. Although the conservation of the Nuage throughout evolution accentuates its essentiality, its role(s) and the exact mechanism(s) by which it functions in the germ line still remain unknown. Here, we report a Nuage component, Krimper (KRIMP), in Drosophila melanogaster and show that it ensures the repression of the selfish genetic elements in the female germ line. The Krimp loss-of-function allele exhibited female sterility, defects in karyosome formation and oocyte polarity, and precocious osk translation. These phenotypes are commonly observed in the other Nuage component mutants, vasa (vas) and maelstrom (mael), and the RNA-silencing component mutants, spindle-E (spn-E) and aubergine (aub), suggesting a shared underlying defect that uses RNA silencing. Moreover, we demonstrated that the localization of the Nuage components depends on both SPN-E and AUB and that the selfish genetic elements were derepressed to different extents in the Nuage component mutants, as well as in aub and armitage (armi) mutants. In the Nuage component mutants, vas, krimp, and mael, the levels of roo, I-element, and HeT-A repeat-associated small interfering RNAs were greatly reduced. Hence, our data suggest that the Nuage functions as a specialized center that protects the genome in the germ-line cells via gene regulation mediated by repeat-associated small interfering RNAs.
Yuko Onohara - One of the best experts on this subject based on the ideXlab platform.
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Argonaute2 Protein in Rat Spermatogenic Cells Is Localized to Nuage Structures and LAMP2-Positive Vesicles Surrounding Chromatoid Bodies.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2016Co-Authors: Yuki Fujii, Hideaki Fujita, Yuko Onohara, Sadaki YokotaAbstract:Localization of Argonaute2 (AGO2) protein--an essential component for the processing of small interfering RNA (siRNA)-directed RNA interference (RNAi) in RNA-induced silencing complex (RISC) in Nuage of rat spermatogenic cells--was evaluated by immunofluorescence microscopy (IFM) and immunoelectron microscopy (IEM). AGO2 was shown, for the first time, to be localized to four previously classified types of Nuage: irregularly shaped perinuclear granules (ISPGs), intermitochondrial cement (IMC), satellite bodies (SBs), and chromatoid bodies (CBs). Dual IEM staining for AGO2/Maelstrom (MAEL) protein or AGO2/MIWI protein demonstrated that AGO2 is colocalized with MAEL or MIWI proteins in these types of Nuage. Dual IFM and IEM staining of AGO2/lysosomal-associated membrane protein 2 (LAMP2) showed that CB in round spermatids are in contact with and surrounded by LAMP2-positive vesicles, whereas Nuage in pachytene spermatocytes are not. Taken together, our findings indicate that: (i) AGO2 in pachytene spermatocytes functions in ISPGs, IMC, and SBs; (ii) AGO2 in round spermatids functions in CBs, and that CBs are associated with lysosomal compartments.
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expression of mael in Nuage and non Nuage compartments of rat spermatogenic cells and colocalization with ddx4 ddx25 and miwi
Histochemistry and Cell Biology, 2013Co-Authors: Miki Takebe, Yuko Onohara, Sadaki YokotaAbstract:The functions of MAELSTROM protein (MAEL) in spermatogenesis are gradually being identified but the precise distribution of MAEL in spermatogenic cells during spermatogenesis has not yet been mapped. We studied the expression of MAEL in rat testis by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence staining showed that MAEL was localized in intermitochondrial cement, irregularly-shaped perinuclear granules and satellite bodies of pachytene spermatocytes, and in chromatoid bodies of spermatids. The SBs appeared exclusively in pachytene spermatocytes at stages IX-X and were stained strongly for MAEL. In step 12-19 spermatids, many granules stained for MAEL but not DDX4. These granules were confirmed to be non-Nuage structures, including mitochondria-associated granules, reticulated body, granulated body by IEM. In the neck region of late spermatids and sperm, MAEL-positive small granules were found. MAEL is colocalized with MIWI in Nuage and non-Nuage. The results suggest that MAEL seems to function in Nuage and non-Nuage structures and interacts with MIWI.
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Nuage components and their contents in mammalian spermatogenic cells as revealed by immunoelectron microscopy
2012Co-Authors: Yuko Onohara, Sadaki YokotaAbstract:The Nuage is a germ cell-specific organelle that has been studied for more than a hundred years. It was first discovered in spermatogenic cells as a perinuclear granule stained by basic dyes and visualized using a light microscope. Morphological studies using electron microscopes demonstrated that ”chromatoid body (CB)” is a Nuage component in spermatids, and that “inter-mitochondrial cement (IMC)” is another Nuage component found in spermatocytes. During meiosis, the IMC disappears, but the CB soon reforms in post-meiotic spermatids. Recent morphological and molecular biological studies have identified many components of Nuage, and suggest that they play roles in the silencing, decay, and storage of RNA, and in the aggresome system. In this report, we summarize recent findings related to Nuage and discuss their functions in spermatogenic cells.
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Expression of DDX25 in Nuage components of mammalian spermatogenic cells: immunofluorescence and immunoelectron microscopic study
Histochemistry and Cell Biology, 2012Co-Authors: Yuko Onohara, Sadaki YokotaAbstract:The localization of DDX25/GRTH and gonadotropin-stimulated RNA helicase was studied in the spermatogenic cells of rat, mouse, and guinea pig by immunofluorescence and immunoelectron microscopy (IEM). Immunofluorescence studies identified four kinds of granular staining: (1) fine particles observed in meiotic cells; (2) small granules associated with a mitochondrial marker, appearing in pachytene spermatocytes after stage V; (3) short strands lacking the mitochondrial marker in late spermatocytes; and, (4) large irregularly shaped granules in round spermatids. IEM identified DDX25 signals in nine compartments: (1) fine dense particles in the meiotic cells; (2) intermitochondrial cement; (3) loose aggregates of 70–90 nm particles; (4) chromatoid bodies; (5) late chromatoid bodies; (6) satellite bodies; (7) granulated bodies; (8) mitochondria-associated granules; and, (9) reticulated bodies. Compartments (1) to (6) were previously classified into Nuage while (7) to (9) were classified as Nuage components by the present study. The results suggest that DDX25 functions in these nine compartments.
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Localization of mouse vasa homolog protein in chromatoid body and related Nuage structures of mammalian spermatogenic cells during spermatogenesis
Histochemistry and Cell Biology, 2010Co-Authors: Yuko Onohara, Toshiyuki Fujiwara, Takanori Yasukochi, Masaru Himeno, Sadaki YokotaAbstract:The localization of vasa homolog protein in the spermatogenic cells of mice, rats, and guinea pigs was studied by immunofluorescence and electron microscopies with the antibody against mouse vasa homolog (MVH) protein. By immunofluorescence microscopy, four types of granular staining patterns were identified: (1) fine particles observed in diplotene and meiotic cells, (2) small granules associated with a mitochondrial marker and appearing in pachytene spermatocytes after stage V, (3) strands lacking the mitochondrial marker in late spermatocytes, and (4) large irregularly shaped granules in round spermatids. Immunoelectron microscopy defined the ultrastructural profiles of these MVH protein-positive granules: the first type consisted of small dense particles, the second had intermitochondrial cement (IMC), the third type, consisting of strands, had loose aggregates of either material dissociated from IMC or 70–90-nm particles, and the fourth had typical chromatoid bodies (CBs). The results suggest that MVH proteins function in these components of Nuage. MVH protein-positive structures other than CBs disappeared during meiosis and CB appeared first in early spermatids. The results suggest that the formation of Nuage is discontinued between spermatocytes and spermatids. The formation of Nuage in spermatocytes and of CB in spermatids is discussed.
Martin M Matzuk - One of the best experts on this subject based on the ideXlab platform.
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the Nuage mediates retrotransposon silencing in mouse primordial ovarian follicles
Development, 2013Co-Authors: Ai Khim Lim, Chanchao Lorthongpanich, Ting Gang Chew, Chin Wee Godwin Tan, Yan Ting Shue, Sathish Balu, Natalia V Gounko, Satomi Kuramochimiyagawa, Martin M MatzukAbstract:Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the Nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of Nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.
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gasz is essential for male meiosis and suppression of retrotransposon expression in the male germline
PLOS Genetics, 2009Co-Authors: Gregory M Buchold, Michael P Greenbaum, Kathleen H Burns, Alan R Harris, Cristian Coarfa, Preethi H Gunaratne, Martin M MatzukAbstract:Nuage are amorphous ultrastructural granules in the cytoplasm of male germ cells as divergent as Drosophila, Xenopus, and Homo sapiens. Most Nuage are cytoplasmic ribonucleoprotein structures implicated in diverse RNA metabolism including the regulation of PIWI-interacting RNA (piRNA) synthesis by the PIWI family (i.e., MILI, MIWI2, and MIWI). MILI is prominent in embryonic and early post-natal germ cells in Nuage also called germinal granules that are often associated with mitochondria and called intermitochondrial cement. We find that GASZ (Germ cell protein with Ankyrin repeats, Sterile alpha motif, and leucine Zipper) co-localizes with MILI in intermitochondrial cement. Knockout of Gasz in mice results in a dramatic downregulation of MILI, and phenocopies the zygotene–pachytene spermatocyte block and male sterility defect observed in MILI null mice. In Gasz null testes, we observe increased hypomethylation and expression of retrotransposons similar to MILI null testes. We also find global shifts in the small RNAome, including down-regulation of repeat-associated, known, and novel piRNAs. These studies provide the first evidence for an essential structural role for GASZ in male fertility and epigenetic and post-transcriptional silencing of retrotransposons by stabilizing MILI in Nuage.
Seokyong Choi - One of the best experts on this subject based on the ideXlab platform.
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pirna associated germline Nuage formation and spermatogenesis require mitopld profusogenic mitochondrial surface lipid signaling
Developmental Cell, 2011Co-Authors: Huiyan Huang, Andrew J Morris, Xiaoxue Peng, Seokyong Choi, Krishna Sarma, Michael A FrohmanAbstract:Summary The mammalian Phospholipase D MitoPLD facilitates mitochondrial fusion by generating the signaling lipid phosphatidic acid (PA). The Drosophila MitoPLD homolog Zucchini (Zuc), a proposed cytoplasmic nuclease, is required for piRNA generation, a critical event in germline development. We show that Zuc localizes to mitochondria and has MitoPLD-like activity. Conversely, MitoPLD −/− mice exhibit the meiotic arrest, DNA damage, and male sterility characteristic of mice lacking piRNAs. The primary function of MitoPLD seems to be the generation of mitochondrial-surface PA. This PA in turn recruits the phosphatase Lipin 1, which converts PA to diacylglycerol and promotes mitochondrial fission, suggesting a mechanism for mitochondrial morphology homeostasis. MitoPLD and Lipin 1 have opposing effects on mitochondria length and on intermitochondrial cement (Nuage), a structure found between aggregated mitochondria that is implicated in piRNA generation. We propose that mitochondrial-surface PA generated by MitoPLD/Zuc recruits or activates Nuage components critical for piRNA production.
