Naomi Nakagata

Kumamoto University, Kumamoto, Kumamoto, Japan

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Publications (153)522.69 Total impact

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    ABSTRACT: Background Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing permits the rapid production of genetically engineered mice. To make the most of this innovative technology, a streamlined procedure is needed for the robust construction of CRISPR/Cas9 vectors, the efficient preparation of mouse oocytes, and refined genotyping strategies. Although we previously demonstrated the applicability of oocyte cryopreservation technologies and various genotyping methods in the production of transcription activator-like effector nuclease-mediated genome editing in mice, it has not yet been clarified whether these techniques can be applied to the CRISPR/Cas9-mediated generation of knockout mice. In this study, we investigated easy, efficient, and robust methods of creating knockout mice using several CRISPR/Cas9 systems. Results We constructed three types of CRISPR/Cas9 vectors expressing: 1) single guide RNA (gRNA) and Cas9 nuclease, 2) two gRNAs and Cas9 nickase, and 3) two gRNAs and FokI-dCas9, targeting the same genomic locus. These vectors were directly microinjected into the pronucleus of freeze-thawed fertilized oocytes, and surviving oocytes were transferred to pseudopregnant ICR mice. Cas9 nuclease resulted in the highest mutation rates with the lowest birth rates, while Cas9 nickase resulted in the highest birth rates with the lowest mutation rates. FokI-dCas9 presented well-balanced mutation and birth rates. Furthermore, we constructed a single all-in-one FokI-dCas9 vector targeting two different genomic loci, and validated its efficacy by blastocyst analysis, resulting in highly efficient simultaneous targeted mutagenesis. Conclusions Our report offers several choices of researcher-friendly consolidated procedures for making CRISPR/Cas9-mediated knockout mice, with sophisticated construction systems for various types of CRISPR vectors, convenient preparation of in vitro fertilized or mated freeze-thawed oocytes, and an efficient method of mutant screening. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0144-x) contains supplementary material, which is available to authorized users.
    BMC Biotechnology 05/2015; 15. DOI:10.1186/s12896-015-0144-x · 2.59 Impact Factor
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    ABSTRACT: GM1-gangliosidosis is an inherited disorder characterized by the accumulation of GM1-gangliosides in many tissues and organs, particularly in the brain. Currently, there is no treatment available for patients with ganglioside storage diseases. Therefore, we investigated the effects of cyclodextrins (CyDs) on the GM1-ganglioside level in EA1 cells, fibroblasts from patients with GM1-gangliosidosis. The concentrations of cholesterol and phospholipids in supernatants were determined by Cholesterol E-test Wako and Phospholipid C-test Wako, respectively. The effects of CyDs on GM1-ganglioside levels in EA1 cells using fluorescence-labelled cholera toxin B-subunit, which can bind to GM1-gangliosides specifically, were investigated by flow cytometry and confocal laser scanning microscopy. The treatment with methylated CyDs, hydroxypropylated CyDs and branched CyDs decreased GM1-ganglioside levels in EA1 cells at 1 mm for 24 h. Unexpectedly, there was no significant change in the efflux of cholesterol or phospholipids from the cells after treatment with CyDs under the same experimental conditions, indicating that the efflux of membrane components is not associated with down-regulation of GM1-ganglioside levels in EA1 cells upon CyDs treatment. CyDs may have the potential as drugs for GM1-gangliosidosis, although the mechanism should be thereafter clarified. © 2015 Royal Pharmaceutical Society.
    Journal of Pharmacy and Pharmacology 04/2015; DOI:10.1111/jphp.12405 · 2.16 Impact Factor
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    ABSTRACT: The developmental processes of the genital tubercle (GT), the anlage of the external genitalia, possess several developmental aspects, including GT outgrowth, urethral tube formation, and epithelial differentiation of the urethra. The GT comprises the mesenchyme derived from the lateral mesoderm, ectodermal epithelium, and endodermal epithelium (embryonic urethral epithelium). The three tissue layers develop the GT coordinately. Around the initial stage of GT outgrowth (E11.5), FGF signaling was detected in the mesenchyme of the GT. FGF signaling was detected in the three tissue layers of the GT around the early stage of urethral formation (E13.5). Subsequently, FGF signaling was predominantly detected in the urethral epithelium (E14.5). Tissue-specific roles of FGF signaling in GT development were revealed by conditional Fgfr gene knockout approaches. Mesenchymal FGF signaling in the early-stage GT is required for its outgrowth. Ectodermal FGF signaling in the GT is required for the differentiation of the ectoderm and urethral epithelium at their junction to form the proper urethral tube. Endodermal FGF signaling in the GT is required for the stratification and cell adhesive characteristics of the urethral epithelium. The current study suggests that spatiotemporally regulated FGF signaling plays tissue-specific roles in multiple processes of external genitalia development. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Developmental Dynamics 03/2015; DOI:10.1002/dvdy.24277 · 2.67 Impact Factor
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    ABSTRACT: Archives of cryopreserved sperm harvested from genetically engineered mice, in mouse resource centers, are a readily accessible genetic resource for the scientific community. We previously reported that exposure of oocytes to reduced glutathione (GSH) greatly improves the fertilization rate of frozen-thawed mouse sperm. Application of GSH to in vitro fertilization techniques is widely accepted as a standard protocol to produce sufficient numbers of mice from cryopreserved sperm. However, the detailed mechanism of the enhancement of fertilization mediated by GSH in vitro is not fully understood. Here we focused on the chemical by determining the effects of its amino acids constituents and cysteine analogs on the fertilization of oocytes by frozen-thawed sperm. Furthermore, we determined the stability of these compounds in aqueous solution. We show here that L-cysteine (L-Cys), D-cysteine (D-Cys), or N-acetyl-L-cysteine (NAC) increased the rate of fertilization when added to the medium but did not adversely affect embryo development in vitro or in vivo. The levels of thiol groups of proteins in the zona pellucida (ZP) and the expansion of the ZP were increased by L-Cys, D-Cys, and NAC. These effects were abrogated by the methylation of the thiol group of L-Cys. NAC was the most stable of these compounds in the fertilization medium at 4°C. These results suggest that the thiol groups of cysteine analogs markedly enhance the fertilization rate of mouse oocytes. Copyright 2015 by The Society for the Study of Reproduction.
    Biology of Reproduction 02/2015; 92(4). DOI:10.1095/biolreprod.114.125443 · 3.45 Impact Factor
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    ABSTRACT: The Wolffian duct (WD) is a primordium of the male reproductive tract and kidney collecting duct system. Fibroblast growth factor receptors (FGFRs), members of the receptor tyrosine kinase (RTK) family, are essential for kidney development. Although the functions of FGFR signaling in kidney morphogenesis have been analyzed, their function in WD development has not been comprehensively investigated. Here, we demonstrate that Fgfr2 is the major Fgfr gene expressed throughout the WD epithelia and that it is essential for the maintenance of the WD, specifically in the caudal part of the WD. Hoxb7-Cre mediated inactivation of Fgfr2 in the mouse WD epithelia resulted in the regression of the caudal part of the WD and abnormal male reproductive tract development. Cell proliferation and expression of the downstream target genes of RTK signaling (Etv4 and Etv5) were decreased in the caudal part of the WD epithelia in the mutant embryos. Cranial (rostral) WD formation and ureteric budding were not affected. Ret, Etv4, and Etv5 expression were sustained in the ureteric bud of the mutant embryos. Taken together, these data suggest region-specific requirements for FGFR2 signaling in the developing caudal WD epithelia. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Developmental Biology 02/2015; 122(1). DOI:10.1016/j.ydbio.2015.01.023 · 3.64 Impact Factor
  • Toru Takeo, Naomi Nakagata
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    ABSTRACT: The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease. Consequently, a huge number of novel mouse models are generated each year to support active research programs. However, it is neither ethically justifiable, nor economically viable to maintain mouse colonies on the shelf that are not part of active research programs. This means that novel mouse lines have to be preserved in some way. If this is not done and the line is simply killed off, the genetics will be lost to future generations of scientists. This article describes the current practices used in cryopreservation laboratories to archive and recover mouse embryos frozen using controlled-rate freezing and vitrification techniques. Copyright © 2014 John Wiley & Sons, Inc.
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    ABSTRACT: SNiemann-Pick disease type C (NPC) is a lysosomal storage disease characterized by abnormal accumulation of free cholesterol and glycolipids. Here, we established induced pluripotent stem cell (iPSC) lines from NPC patients. Hepatocyte-like cells (HLCs) and neural progenitors derived from the iPSC lines accumulated cholesterol and displayed impaired autophagy and ATP production. A molecular signature related to lipid metabolism was also impaired in the NPC-iPSC-derived HLCs. These findings indicate that iPSC-derived cells can phenocopy human NPC. We also newly found that 2-hydroxypropyl-γ-cyclodextrin (HPGCD) could reduce the cholesterol accumulation and restore the functional and molecular abnormalities in the NPC patient-derived cells, and do so more effectively than 2-hydroxypropyl-β-cyclodextrin treatment. In addition, NPC model mice showed an improved liver status and prolonged survival with HPGCDs. Thus, iPSC lines derived from patient cells are powerful tools to study cellular models of NPC, and HPGCD is a potential new drug candidate for future treatment of this disease. This article is protected by copyright. All rights reserved.
    Stem Cells 12/2014; 33(4). DOI:10.1002/stem.1917 · 7.70 Impact Factor
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    ABSTRACT: Hyaluronidase is generally used to remove cumulus cells from mouse oocytes before oocyte cryopreservation, intracytoplasmic sperm injection or DNA injection. In general, use of cumulus-free mouse oocytes decreases in vitro fertilizing ability compared with cumulus-surrounded oocytes. The effect of hyaluronidase exposure on the quality of mouse oocytes is not fully understood. Here, we investigated the effect of hyaluronidase exposure time on the fertilization rate of fresh and vitrified mouse oocytes and their subsequent developmental ability in vitro. We found that the fertilization rate decreased with hyaluronidase treatments. This reduction in the fertilization rate following treatment with hyaluronidase was fully reversed by removal of the zona pellucida. In addition, oocytes treated with hyaluronidase for 5 min or longer had a reduced capacity to develop to the morula and blastocyst stage. The survival, fertilization, and developmental rates of vitrified-warmed oocytes were also reduced by longer exposure to hyaluronidase. In conclusion, these results suggest that prolonged exposure to hyaluronidase decreases the quality of mouse oocytes and shorter hyaluronidase treatment times may help achieve a stable and high fertilization rate in fresh and cryopreserved oocytes.
    Journal of Reproduction and Development 12/2014; 60(6). DOI:10.1262/jrd.2014-045 · 1.64 Impact Factor
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    ABSTRACT: Masculinization of external genitalia is an essential process in the formation of the male reproductive system. Prominent characteristics of this masculinization are the organ size and the sexual differentiation of the urethra. Although androgen is a pivotal inducer of the masculinization, the regulatory mechanism under the control of androgen is still unknown. Here, we address this longstanding question about how androgen induces masculinization of the embryonic external genitalia through the identification of the v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (Mafb) gene. Mafb is expressed prominently in the mesenchyme of male genital tubercle (GT), the anlage of external genitalia. MAFB expression is rarely detected in the mesenchyme of female GTs. However, exposure to exogenous androgen induces its mesenchymal expression in female GTs. Furthermore, MAFB expression is prominently down-regulated in male GTs of androgen receptor (Ar) KO mice, indicating that AR signaling is necessary for its expression. It is revealed that Mafb KO male GTs exhibit defective embryonic urethral formation, giving insight into the common human congenital anomaly hypospadias. However, the size of Mafb KO male GTs is similar with that of wild-type males. Moreover, androgen treatment fails to induce urethral masculinization of the GTs in Mafb KO mice. The current results provide evidence that Mafb is an androgen-inducible, sexually dimorphic regulator of embryonic urethral masculinization.
    Proceedings of the National Academy of Sciences 10/2014; DOI:10.1073/pnas.1413273111 · 9.81 Impact Factor
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    ABSTRACT: The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer’s disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.
    Stem Cell Research 10/2014; 13(3). DOI:10.1016/j.scr.2014.10.001 · 3.91 Impact Factor
  • 12th Transgenic Technology Meeting, Edinburgh; 10/2014
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    ABSTRACT: Each year, thousands of new mouse models are generated around the world to further biomedical research. Unfortunately, the cost of maintaining mouse colonies makes it uneconomical to keep strains on the shelf that are not part of active research programs. Ideally, these retired strains should be archived. If this is not done and the line is simply killed off, the genetics are lost to future generations of scientists. Traditionally, embryo freezing has been used to cryopreserve mice, but this is expensive, time consuming, requires large numbers of donor females, and usually involves invasive superovulation procedures. Sperm freezing circumvents all of these disadvantages and is rapidly becoming the technique of choice for many repositories. This has been made possible through the use of refined cryoprotective agents and the development of improved in vitro fertilization techniques. This article describes two popular sperm freezing techniques employed by mouse repositories to archive spermatozoa using cryoprotective agents supplemented with either L-glutamine or monothioglycerol. Copyright © 2014 John Wiley & Sons, Inc.
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    ABSTRACT: The 21st century has seen a huge proliferation in the availability of genetically altered mice. The availability of these resources has been accompanied by ever greater opportunities for international collaborations between laboratories involving the exchange of mouse strains. This exchange can involve significant costs in terms of animal welfare and transportation expenses. In an attempt to mitigate some of these costs, the mouse community has developed a battery of techniques that can be used to avoid transporting live mice. Transporting frozen embryos and sperm at liquid nitrogen (LN2 ) temperatures using dry shippers has been common practice for some time. However, current advances in this field have refined transportation procedures and introduced new techniques for disseminating embryos and sperm: for example, shipping frozen sperm on dry ice, exchanging unfrozen epididymides from which sperm can be extracted, and transporting frozen/thawed embryos in isotonic media. This article discusses some of the current practices used by laboratories to transport mouse strains around the world without having to exchange live mice. Copyright © 2014 John Wiley & Sons, Inc.
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    ABSTRACT: Historically, timed mating of either naturally cycling or superovulated females has been the mainstay of pre-implantation embryo production. However, rising cage costs and the rapid expansion of biomedical research programs has necessitated the development of high-throughput approaches to mouse embryo production. In vitro fertilization (IVF) represents one such versatile tool offering many advantages to busy mouse facilities in terms of efficient use of space and resources. For example, strains can be taken off the shelf, frozen quickly as sperm, and recovered at a later date, small colonies can be rapidly expanded to meet demand, and IVF can be used to rescue strains that fail to breed or where the founder male is ill or has died suddenly. This article describes an IVF protocol currently used by many reproductive technologists to assist mouse biology programs. Copyright © 2014 John Wiley & Sons, Inc.
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    ABSTRACT: Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.
    Development 05/2014; 141(10):2157-64. DOI:10.1242/dev.105148 · 6.27 Impact Factor
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    ABSTRACT: The BC (bulbocavernosus) is a sexually dimorphic muscle observed only in males. ARKO (Androgen receptor knock out) mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at E16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signalling in this process, muscle-specific androgen receptor (AR) mutation was introduced which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as MyoD, Myog, and Pax7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the non-myocytic cells utilizing Sall1 (spalt-like transcription factor 1) Cre driver mouse was performed which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the SallAR (Sall1 Cre:AR(L-/Y)) mutant embryos and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by Cdk-inhibitors. An increased expression of p21 was observed in the BC myoblast of the SallAR mutant and WT female. Altogether, this study suggets that the non-myocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.
    Endocrinology 04/2014; DOI:10.1210/en.2014-1008 · 4.64 Impact Factor
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    ABSTRACT: Prostate is a male sex accessory organ. The prostatic epithelia consist primarily of basal and luminal cells that differentiate from embryonic urogenital sinus epithelia. Prostate tumors are believed to originate in the basal and luminal cells. However, factors that promote normal epithelial differentiation have not been well elucidated, particularly for bone morphogenetic protein (Bmp) signaling. This study shows that Bmp signaling prominently increases during prostatic differentiation in the luminal epithelia, which is monitored by the expression of pSmad1/5/8. To elucidate the mechanism of epithelial differentiation and the function of Bmp signaling during prostatic development, conditional male mutant mouse analysis for the epithelial-specific Bmp receptor 1a (Bmpr1a) was performed. We demonstrate that Bmp signaling is indispensable for luminal cell maturation, which regulates basal cell proliferation. Expression of the prostatic epithelial regulatory gene Nkx3.1 was significantly reduced in the Bmpr1a mutants. These results indicate that Bmp signaling is a key factor for prostatic epithelial differentiation, possibly by controlling the prostatic regulatory gene Nkx3.1.
    Endocrinology 04/2014; 155(7):en20132054. DOI:10.1210/en.2013-2054 · 4.64 Impact Factor
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    ABSTRACT: The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh(CreERT2)). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh(CreERT2/+); β-catenin(flox(ex3)/+); BmprIA(flox/-) mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.Cell Death and Differentiation advance online publication, 14 March 2014; doi:10.1038/cdd.2014.21.
    Cell death and differentiation 03/2014; 21(6). DOI:10.1038/cdd.2014.21 · 8.39 Impact Factor
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    ABSTRACT: Sperm cryopreservation has been widely adopted for maintenance of the genetically engineered mouse (GEM). The cryopreserved sperm are being exchanged among many institutes worldwide. However, the recipients are not always able to obtain high fertilization rates with the frozen sperm shipped from senders. In this study, we cryopreserved mouse sperm via various methods and performed in vitro fertilization (IVF) in which the combination of methyl-beta-cyclodextrin for sperm preincubation and reduced glutathione for insemination was used (the MBCD-GSH IVF). In addition, frozen sperm sent from the Jackson Laboratory (USA) were thawed and used for IVF in the same manner. The fertilization rates of both the sperm cryopreserved via the methods applied in some countries and the cryopreserved GEM sperm improved when used with the MBCD-GSH IVF method. Therefore, we strongly believe that the MBCD-GSH IVF method brings about relatively high fertilization rates with any strain of frozen mouse sperm.
    Journal of Reproduction and Development 02/2014; 60(2). DOI:10.1262/jrd.2013-141 · 1.64 Impact Factor

Publication Stats

3k Citations
522.69 Total Impact Points


  • 2000–2015
    • Kumamoto University
      • • Department of Reproductive Engineering
      • • Institute of Resource Development and Analysis (IRDA)
      • • Center for Animal Resources and Development
      • • Department of Developmental Genetics
      Kumamoto, Kumamoto, Japan
  • 2007
    • University of Utah
      • Department of Pediatrics
      Salt Lake City, Utah, United States
  • 2005
    • Keio University
      • School of Medicine
      Tokyo, Tokyo-to, Japan
  • 2003
    • Yamagata University
      • Laboratory Animal Center
      Ямагата, Yamagata, Japan
    • Centre for Water Resources Development and Management
      Kunnamangalam, Kerala, India
  • 2002
    • University of Texas at Dallas
      Richardson, Texas, United States
  • 1997
    • The University of Tokyo
      • Institute of Medical Science
      Tokyo, Tokyo-to, Japan