記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/glia

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1126

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

その他リンク： https://syndication.highwire.org/content/doi/10.1530/REP-18-0043

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：日本語   出版者・発行元：(一社)日本卵子学会  

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-017-18365-z

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   出版者・発行元：NATL ACAD SCIENCES  

DOI： 10.1073/pnas.1711468114

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語  

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語  

記述言語：英語  

記述言語：英語  

記述言語：英語  

記述言語：英語  

記述言語：英語  

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語  

記述言語：英語  

記述言語：日本語  

J-GLOBAL

記述言語：日本語  

J-GLOBAL

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：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

Web of Science

PubMed

記述言語：日本語  

CiNii Books

記述言語：日本語  

J-GLOBAL

担当区分：筆頭著者   記述言語：英語  

記述言語：英語  

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】哺乳類の初期胚は胚盤胞期において細胞分化が明確化する。すなわち，将来胎子組織となる内部細胞塊(ICM)と，胚体外組織を形成する栄養外胚葉(TE)の2種の細胞群に分化し，それぞれの細胞に特有の遺伝子発現を示すようになる。これまでに我々は，ウシ胚盤胞期胚においてICMあるいはTE特異的な発現遺伝子パターンを明らかにするために部位特異的発現を示す遺伝子群をマイクロアレイにより決定した。本研究では，これらの遺伝子群のうち，定量PCRおよびIn situ hybridization法によってTE特異的な発現が新たに確認されたConnective tissue growth factor (CTGF )について着目し，初期胚における発現動態を解析した。体細胞の研究において，CTGFはCDX2の上流で機能するTEAD4の標的遺伝子であると考えられているが，ウシ胚での発現の挙動は明らかになっていない。そこで，TEAD4に関しても発生過程における発現動態を解析した。【方法】屠場由来の卵巣より採取した卵子を体外成熟，受精，培養に供した。体外受精後の発生胚を8–16細胞期，桑実期，胚盤胞期の3ステージに分け，1ユニット10個として各時期で3ユニットずつサンプリングした。各サンプル胚からRNAを抽出後，cDNAを合成し，定量PCRに供してmRNA発現レベルの比較を行った。【結果および考察】CTGFは8–16細胞期から拡張胚盤胞期にかけて約10倍に発現レベルが有意に上昇した(P<0.01)。一方，TEAD4についても同様に8–16細胞期から胚盤胞期にかけて約20倍に発現レベルが上昇し，有意差が認められた(P<0.01)。CTGFおよびTEAD4遺伝子ともに，8–16細胞期から胚盤胞期にかけて発現レベルが上昇したことから，胚盤胞期胚でのCTGFのTE細胞特異的発現にTEAD4が関連していることが示唆された。

DOI： 10.14882/jrds.106.0.P-82.0

J-GLOBAL

記述言語：日本語  

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

ウシ胚盤胞期胚におけるTEAD4およびYAPの発現動態に関する研究<br><br>○加川真二朗，長友啓明，高橋昌志，川原学 (北大院農)<br><br>【目的】哺乳類の個体発生における最初の分化は胚盤胞期胚に起こり，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。この分化調節機構についてマウス胚では解析が進んでおり，Hippo pathway下流の転写コアクチベーターであるYes-associated protein(Yap)が重要な役割を果たすことがわかっている。すなわち，胚のそれぞれの割球における転写因子Tead4の細胞内局在を，Yapが直接変化させることにより分化を制御している。しかし，ウシ胚でも同一の仕組みで分化が制御されているかは不明である。そこで，ウシ胚におけるICM/TE分化制御機構を探るため，ウシTEAD4(bTEAD4)遺伝子の胚盤胞期胚における発現解析を行い，さらにウシYAP(bYAP)遺伝子の塩基配列を解析した。【方法】体外受精により作出したウシ胚盤胞期胚からICMおよびTEの部分胚を採取した。これらのサンプルに通常の全体胚(Whole)を加え，3種のサンプルより抽出したtotal RNAを用いて，bTEAD4の定量PCRを行った。また，RT-PCRでbYAP遺伝子cDNAをクローニングし塩基配列を決定したのち，マウスYap(mYap)のデータベースと比較した。【結果および考察】ICM，TE，Whole胚でのbTEAD4の発現を定量PCRで比較した結果，TEでの発現レベルがICMおよびWholeサンプルの発現レベルよりも高い値を示した。また，決定したbYAP塩基配列からタンパク質一次構造を予測しマウスと比較した結果，ウシではN末端領域が欠損していることが判明した。この欠損領域はTeadファミリー転写因子との結合領域であり，mYapタンパク質局在に重要な役割を果たすリン酸化部位も含まれている。以上の結果から，ウシ胚では，マウス胚のようなTead-Yapの直接的な相互作用を介した機構とは異なる様式でICM/TEの細胞分化が制御されている可能性が示された。

DOI： 10.14882/jrds.105.0_106

J-GLOBAL

記述言語：日本語  

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】胚盤胞期胚は，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。さらに，TEはICMへの隣接の有無から極栄養外胚葉(pTE)と壁栄養外胚葉(mTE)に分類される。ウシ胚では，これらの細胞での遺伝子発現プロファイルは十分に理解されていない。第103会大会では全体胚(whole)，および，顕微操作により機械的に分断した部分胚(ICM側: ICM + pTE; TE側: mTE)の解析結果を報告した。しかし、ICM単体のサンプルでの解析が欠けていたため，今回はTEを完全に除去したICMのサンプルを採取して遺伝子発現解析を追加した。さらに，ICM，TEサンプル間で発現差のあった遺伝子についてリアルタイムPCRを行った。【方法】ウシ胚盤胞期胚TEを界面活性剤で除去したICMサンプルを使用し、定法により調整してマイクロアレイに供した。解析結果を，主成分分析やオントロジー分析を行い、各サンプル間での遺伝子発現パターンの相違を調べ，発現差のみられた肝細胞核因子4A(HNF4A)などの遺伝子についてリアルタイムPCRを行った。【結果】マイクロアレイの結果，主成分分析により遺伝子発現パターンを比較したところ，4種類の部分胚，すなわちmTE，whole，ICM+pTE，ICMにおいてそれぞれ特異的な遺伝子発現パターンを示した。とくに，mTEとICMの発現パターンが大きく異なっていた。また、pTEとmTEを比較すると、pTEで発現が顕著に上昇している遺伝子は3遺伝子のみであった。ICMとmTEにおいて，それぞれ特異的に発現レベルが有意に上昇していた遺伝子について成長因子や転写因子の機能的な側面から15個ずつ選出した。ICMで発現が上昇していた遺伝子中には，HNF4Aのような転写因子が含まれており，mTEと比較して17倍以上の差がみられた。同様の動向を示す遺伝子も含めて，リアルタイムPCRによる発現解析も行った。今回、ウシ胚盤胞期胚で部位毎に分別したサンプルを用い、ICMとTEで発現差のみられた遺伝子群から、機能的側面も考慮し、候補遺伝子を絞り込んだ。今後更なる解析によりウシ胚盤胞期胚における分化関連遺伝子探索に役立てたい。

DOI： 10.14882/jrds.104.0.1062.0

J-GLOBAL

記述言語：日本語   出版者・発行元：日本繁殖生物学会  

【目的】胚盤胞期胚は，構造的に内部細胞塊(ICM)と栄養外胚葉(TE)に二分される。さらに，TEはICMへの隣接の有無から極栄養外胚葉(pTE)と壁栄養外胚葉(mTE)に分類される。ウシ胚では，これらの細胞での遺伝子発現プロファイルは十分に理解されていない。そこで本研究では，ウシ胚盤胞期胚の部位ごとに細胞を分別してマイクロアレイ法による網羅的遺伝子発現解析を行った。【方法】マイクロアレイ解析には灌流採取した体内由来胚(vivo胚)，体外受精由来胚(vitro胚)，体細胞クローン胚(SCNT胚)の3種類について，透明帯を除去した全体胚，および，ICM側(ICM + pTE)とTE側(mTE)を顕微操作により機械的に分断した部分胚を使用した。定法により調整したサンプルをマイクロアレイにかけて，主成分分析(PCA)や解析サイトFatiGOでのオントロジー分析を行い各サンプル間での遺伝子発現パターンの相違を調べた。【結果】マイクロアレイの結果，PCAにより遺伝子発現パターンを俯瞰したところ，それぞれの部分胚においてvivoおよびvitro胚由来では近似した発現パターンを示した。しかし，SCNT胚由来ではそれらとは異なる発現パターンを示し，この傾向はオントロジー分析の結果でも同様であった。ICM側およびTE側部分胚で有意に発現レベルが異なっていた遺伝子を調べたところ，vivo胚で984個，vitro胚で2279個，SCNT胚では2599個であった。これらの結果から，胚盤胞期胚においてICM側とTE側の部分胚を使ったマイクロアレイによる網羅的遺伝子発現解析により部位特異的に発現する遺伝子群を探索できる可能性が示された。

DOI： 10.14882/jrds.103.0.29.0

J-GLOBAL