Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/glia

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1126

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Histone lysine-to-methionine (K-to-M) mutations have been identified as driver mutations in human cancers. Interestingly, these 'oncohistone' mutations inhibit the activity of histone methyltransferases. Therefore, they can potentially be used as versatile tools to investigate the roles of histone modifications. In this study, we generated a genetically engineered mouse line in which an H3.3K36M mutation could be induced in the endogenous H3f3b gene. Since H3.3K36M has been identified as a causative mutation of human chondroblastoma, we induced this mutation in the chondrocyte lineage in mouse embryonic limbs. We found that H3.3K36M causes a global reduction in H3K36me2 and defects in chondrocyte differentiation. Importantly, the reduction of H3K36me2 was accompanied by a collapse of normal H3K27me3 distribution. Furthermore, the changes in H3K27me3, especially the loss of H3K27me3 at gene regulatory elements, were associated with the mis-regulated expression of a set of genes important for limb development, including HoxA cluster genes. Thus, through the in vivo induction of the H3.3K36M mutation, we reveal the importance of maintaining the balance between H3K36me2 and H3K27me3 during chondrocyte differentiation and limb development.

DOI： 10.1080/15592294.2020.1841873

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Motile cilia and flagella require ATP for their formation and function. Although glycolytic enzymes are components of flagellar proteomes, how they translocate to flagella is unknown. Here we show that the expression pattern of the functionally nonannotated gene 4833427G06Rik (C11orf88), which is found only in vertebrates and is designated here as Hoatzin (Hoatz), suggests a functional association of its product with motile cilia and flagella. Hoatz knockout (KO) mice developed hydrocephalus and male infertility in an autosomal recessive manner, and the ependymal cilia frequently showed disorganized axonemes, reducing motility associated with collapsed spermatid flagella during cytodifferentiation. HOATZ was associated with certain proteins, including the flagellar glycolytic enzyme ENO4. In the testes of the Hoatz KO mice, the immature form of ENO4 accumulated in abnormal cytoplasmic puncta of developing spermatids. These data indicate that HOATZ is required for motile ciliogenesis and flagellar genesis in vertebrates by mediating the maturation of ENO4.

DOI： 10.1016/j.isci.2020.100992

PubMed

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.

DOI： 10.1182/blood-2017-12-823369

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bioscientifica  

The first segregation at the blastocyst stage is the symmetry-breaking event to characterize two cell components; namely, inner cell mass (ICM) and trophectoderm (TE). TEA domain transcription factor 4 (TEAD4) is a well-known regulator to determine TE properties of blastomeres in rodent models. However, the roles of bovine TEAD4 in blastocyst development have been unclear. We here aimed to clarify the mechanisms underlining TE characterization by TEAD4 in bovine blastocysts. We first found that the<italic>TEAD4</italic>mRNA expression level was greater in TE than in ICM, which was further supported by TEAD4 immunofluorescent staining. Subsequently, we examined the expression patterns of TE-expressed genes;<italic>CDX2</italic>,<italic>GATA2</italic>and<italic>CCN2</italic>, in the<italic>TEAD4</italic>-knockdown (KD) blastocysts. These expression levels significantly decreased in the<italic>TEAD4</italic>KD blastocysts compared with controls. Of these downregulated genes, the<italic>CCN2</italic>expression level decreased the most. We further analyzed the expression levels of TE-expressed genes;<italic>CDX2</italic>,<italic>GATA2</italic>and<italic>TEAD4</italic>in the<italic>CCN2</italic>KD blastocysts. Strikingly, the<italic>CCN2</italic>KD blastocysts showed the downregulation of<italic>CDX2</italic>,<italic>GATA2</italic>and<italic>TEAD4</italic>. Furthermore, the ratio of TE-to-ICM cell numbers in the<italic>CCN2</italic>KD blastocysts significantly decreased compared to controls. To our knowledge, this is the first study showing the regulation of<italic>CCN2</italic>expression thorough<italic>TEAD4</italic>in mammalian embryos. Not only that, this study also provides evidence that reciprocal regulation of<italic>TEAD4</italic>and<italic>CCN2</italic>is required for TE development with appropriate gene expression in bovine blastocysts.

DOI： 10.1530/rep-18-0043

PubMed

Other Link： https://syndication.highwire.org/content/doi/10.1530/REP-18-0043

Language：English   Publishing type：Research paper (scientific journal)  

Language：Japanese   Publisher：(一社)日本卵子学会  

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Oocytes without a zona pellucida (ZP) often occur as a result of congenital or operational effects, but they are difficult to handle, and embryonic survival is low. Such zona-free (ZF) oocytes are therefore not used in clinics or laboratories. Furthermore, in the laboratory, removal of the ZP is often necessary for genetic manipulation by viral infection or twin production by blastomere separation, but adverse effects on development have been reported. It would therefore be extremely valuable if the embryo could be covered with a structure similar to that of the ZP. In this study, we sought to determine whether an agarose capsule could serve as a substitute for the ZP. Our results indicate that embryos derived from these agarose capsules were able to develop normally, and could be transplanted to obtain viable offspring, without having to remove the agarose capsule. Furthermore, before compaction, the agarose capsule embryos exhibited good freezing tolerance, and survival rate was extremely high compared to ZF embryos. Thus, agarose capsules represent a valuable tool for utilizing oocytes and embryos that lack a ZP, both in a clinical and livestock setting.

DOI： 10.1038/s41598-017-18365-z

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOSCIENTIFICA LTD  

Recently, it has become possible to generate cloned mice using a somatic cell nucleus derived from not only F1 strains but also inbred strains. However, to date, all cloned mice have been generated using F1 mouse oocytes as the recipient cytoplasm. Here, we attempted to generate cloned mice from oocytes derived from the ICR-outbred mouse strain. Cumulus cell nuclei derived from BDF1 and ICR mouse strains were injected into enucleated oocytes of both strains to create four groups. Subsequently, the quality and developmental potential of the cloned embryos were examined. ICR oocytes were more susceptible to damage associated with nuclear injection than BDF1 oocytes, but their activation rate and several epigenetic markers of reconstructed cloned oocytes/ embryos were similar to those of BDF1 oocytes. When cloned embryos were cultured for up to 4 days, those derived from ICR oocytes demonstrated a significantly decreased rate of development to the blastocyst stage, irrespective of the nuclear donor mouse strain. However, when cloned embryos derived from ICR oocytes were transferred to female recipients at the two-cell stage, healthy cloned offspring were obtained at a success rate similar to that using BDF1 oocytes. The ICR mouse strain is very popular for biological research and less expensive to establish than most other strains. Thus, the results of this study should promote the study of nuclear reprogramming not only by reducing the cost of experiments but also by allowing us to study the effect of oocyte cytoplasm by comparing it between strains.

DOI： 10.1530/REP-17-0372

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PubMed

Language：English   Publisher：NATL ACAD SCIENCES  

DOI： 10.1073/pnas.1711468114

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes. Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95 degrees C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Next-generation sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.

DOI： 10.1073/pnas.1701425114

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells2017;35:1189-1196

DOI： 10.1002/stem.2601

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Language：English  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT, INC  

Although animal cloning is becoming increasingly practicable, cloned embryos possess many abnormalities and so there has been a low success rate for producing live animals. This is most likely due to incomplete reprogramming of somatic cell nuclei before they start to develop as the donor nuclei are usually only exposed to the oocyte cytoplasm for 1-2 hours before reconstructed oocytes are activated to avoid oocyte aging. Therefore, in this study, we attempted to extend the exposure period of somatic cell nuclei to the oocyte cytoplasm to determine whether this could enhance reprogramming of donor nuclei. Donor nuclei were transferred into oocytes, following which pseudo-MII spindles (pMIIs) derived from these were extracted and injected into newly collected enucleated oocytes 24 hours after the first nuclear transfer (NT). These serial NT oocytes were then activated and their developmental potential was examined to full term. There was no obvious difference in the pMIIs of reconstructed oocytes at 6 and 24 hours after donor nucleus injection; however, in both of these, the chromosomes were more widely spread inside the spindle than in fresh intact oocytes. Furthermore, a few chromosomes remained in 25% and 47% of these enucleated oocytes at 6 and 24 hours after donor nucleus injection, respectively. When these pMIIs were injected into fresh enucleated oocytes, the developmental rate to blastocyst was significantly lower, but we could still obtain several healthy cloned offspring. Thus, serial NT at intervals of 24 hours using fresh oocytes is possible, but the success rate could not be improved due to loss of chromosomes during the second NT.

DOI： 10.1089/cell.2016.0026

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Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

A common process during preimplantation mammalian development is blastocyst formation, which utilizes signaling through fibroblast growth factor receptor 2 (FGFR2), yet the mechanisms through which FGFR2 signaling affect preimplantation development in bovine embryos remain incompletely understood. Here, we used RNA-interference to investigate the in vitro development, the frequency of blastomere apoptosis, and the mRNA expression of developmental marker genes in FGF receptor 2-knockdown (FGFR2-KD) bovine embryos. A reduction in FGFR2 mRNA did not affect preimplantation development or the frequency of apoptotic blastomeres, but did enhanced proliferation of the inner cell mass in blastocysts (P&lt; 0.05) -which differs from the phenotype reported for bovine embryos using a pharmacological approach (treatment with the pan-FGFR blocker PD173074), but agrees with previous results obtained using mouse embryos. Moreover, the expression of an epiblast marker gene, NANOG, and a primitive endoderm marker gene, GATA6, remained unchanged, whereas the expression of another primitive endoderm marker gene, HNF4A, was significantly reduced in FGFR2-KD embryos. Therefore, FGFR2 signaling appears to be associated with the regulation of inner cell mass development and proliferation during blastocyst formation in cattle.

DOI： 10.1002/mrd.22646

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Cloning animals by nuclear transfer provides the opportunity to preserve endangered mammalian species. However, there are risks associated with the collection of donor cells from the body such as accidental injury to or death of the animal. Here, we report the production of cloned mice from urine-derived cells collected noninvasively. Most of the urine-derived cells survived and were available as donors for nuclear transfer without any pretreatment. After nuclear transfer, 38-77% of the reconstructed embryos developed to the morula/blastocyst, in which the cell numbers in the inner cell mass and trophectoderm were similar to those of controls. Male and female cloned mice were delivered from cloned embryos transferred to recipient females, and these cloned animals grew to adulthood and delivered pups naturally when mated with each other. The results suggest that these cloned mice had normal fertility. In additional experiments, 26 nuclear transfer embryonic stem cell lines were established from 108 cloned blastocysts derived from four mouse strains including inbreds and F1 hybrids with relatively high success rates. Thus, cells derived from urine, which can be collected noninvasively, may be used in the rescue of endangered mammalian species by using nuclear transfer without causing injury to the animal.

DOI： 10.1038/srep23808

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PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Nuclear autoantigenic sperm protein (NASP) is associated with DNA replication, cell proliferation, and cell cycle progression through its specific binding to histones. The aim of this study was to examine the roles of NASP in bovine preimplantation embryonic development. Using NASP gene knockdown (KD), we confirmed the reduction of NASP messenger RNA (mRNA) expression during preimplantation development. NASP KD did not affect cleavage but significantly decreased development of embryos into the blastocyst stage. Furthermore, blastocyst hatching was significantly decreased in NASP KD embryos. Cell numbers in the inner cell mass of NASP KD blastocysts were also decreased compared to those of controls. These results suggest that NASP mRNA expression is required for preimplantation development into the blastocyst stage in cattle.

DOI： 10.1111/asj.12538

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Language：English  

Language：English  

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J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：HOKKAIDO UNIV  

There has been no work on spatiotemporal transcriptomic differences of blastocysts using in vivo- and in vitro-derived, and somatic cell nuclear transfer (SCNT) embryos. Here, we first compared the lineage-differentially transcriptomic profiles of in vivo- and in vitro-derived embryos by microarray analysis using divided into inner cell mass (ICM)-and trophectoderm (TE)-side samples, as well as those derived from SCNT in order to explore lineage-differentially expressed genes that are associated with preimplantation development in cattle. The transcriptomic profiles of the ICM-specific and TE-specific genes were similar between in vitro-derived embryos and in vivo-derived embryos, whereas SCNT embryos exhibited unusual lineage-differentially gene expression regulation at the blastocyst stage. The genes expressed in a spatiotemporal manner between developmentally normal in-vivo derived blastocysts and developmentally abnormal SCNT blastocysts might play critical roles for preimplantation development. Comparing spatial expression dynamics of bovine blastocyst under three different procedures revealed that CHTA was expressed in ICM-side samples of all the embryo types. CIITA is known as the master regulator of major histocompatibility complexes (MHC) class II genes that express in antigen-presenting cells but its biological function in preimplantation embryo is still unknown in mammals. Knockdown of CIITA expression in in vitro-derived embryos did not affect cleavage, but disrupted development of embryos into the blastocyst stage. These findings provide the novel transcriptomic information on blastocyst formation, raising the possibility that immune function-related gene directly plays important roles in bovine preimplantation development.

DOI： 10.14943/jjvr.63.4.159

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Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

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J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable'' for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos

DOI： 10.1095/biolreprod.114.123455

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Language：English  

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J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC STUDY REPRODUCTION  

Mice and cattle use distinct pathways for the first cell segregation into inner cell mass (ICM) and trophectoderm (TE) lineages at the blastocyst stage. However, limited knowledge is available regarding the reliable transcriptional networks that orchestrate the complex developmental processes at this stage in nonrodent species. In order to elucidate the site-dominant transcriptomic properties of bovine blastocysts, we separated cell samples into the ICM and TE using both mechanical and chemical methods and performed in silico prescreening for candidate genes that were site-dominantly expressed in bovine blastocysts. We further performed quantitative real-time PCR and in situ hybridization using the site-specific cell samples. As a result, we identified seven ICM-dominant genes and five TE-dominant genes not found in earlier studies. Our findings provide novel insights into the mechanism of cell-fate specification in the pre-implantation bovine embryo.

DOI： 10.1095/biolreprod.113.108993

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 +/- 2.1% vs. 65.0 +/- 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2013.02.009

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Language：Japanese  

CiNii Books

Language：Japanese  

J-GLOBAL

Authorship：Lead author   Language：English  

Language：English  

Language：Japanese  

DOI： 10.14882/jrds.106.0.P-82.0

J-GLOBAL

Language：Japanese  

Language：Japanese   Publisher：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

DOI： 10.14882/jrds.105.0_106

J-GLOBAL

Language：Japanese  

Language：Japanese   Publisher：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

DOI： 10.14882/jrds.104.0.1062.0

J-GLOBAL

Language：Japanese   Publisher：THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT  

DOI： 10.14882/jrds.103.0.29.0

J-GLOBAL