Publications by authors named "Shoma Nakagawa"

12 Publications

  • Page 1 of 1

Controlled ploidy reduction of pluripotent 4n cells generates 2n cells during mouse embryo development.

Sci Adv 2019 10 16;5(10):eaax4199. Epub 2019 Oct 16.

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, 08003 Barcelona, Spain.

Cells with high ploidy content are common in mammalian extraembryonic and adult tissues. Cell-to-cell fusion generates polyploid cells during mammalian development and tissue regeneration. However, whether increased ploidy can be occasionally tolerated in embryonic lineages still remains largely unknown. Here, we show that pluripotent, fusion-derived tetraploid cells, when injected in a recipient mouse blastocyst, can generate diploid cells upon ploidy reduction. The generated diploid cells form part of the adult tissues in mouse chimeras. Parental chromosomes in pluripotent tetraploid cells are segregated through tripolar mitosis both randomly and nonrandomly and without aneuploidy. Tetraploid-derived diploid cells show a differentiated phenotype. Overall, we discovered an unexpected process of controlled genome reduction in pluripotent tetraploid cells. This mechanism can ultimately generate diploid cells during mouse embryo development and should also be considered for cell fusion-mediated tissue regeneration approaches.
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http://dx.doi.org/10.1126/sciadv.aax4199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795515PMC
October 2019

Ultrafast imaging of cell elasticity with optical microelastography.

Proc Natl Acad Sci U S A 2018 01 16;115(5):861-866. Epub 2018 Jan 16.

Laboratory of Biorheology and Medical Ultrasonics, University of Montreal Hospital Research Center, Montreal, QC, Canada H2X 0A9;

Elasticity is a fundamental cellular property that is related to the anatomy, functionality, and pathological state of cells and tissues. However, current techniques based on cell deformation, atomic force microscopy, or Brillouin scattering are rather slow and do not always accurately represent cell elasticity. Here, we have developed an alternative technique by applying shear wave elastography to the micrometer scale. Elastic waves were mechanically induced in live mammalian oocytes using a vibrating micropipette. These audible frequency waves were observed optically at 200,000 frames per second and tracked with an optical flow algorithm. Whole-cell elasticity was then mapped using an elastography method inspired by the seismology field. Using this approach we show that the elasticity of mouse oocytes is decreased when the oocyte cytoskeleton is disrupted with cytochalasin B. The technique is fast (less than 1 ms for data acquisition), precise (spatial resolution of a few micrometers), able to map internal cell structures, and robust and thus represents a tractable option for interrogating biomechanical properties of diverse cell types.
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http://dx.doi.org/10.1073/pnas.1713395115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798341PMC
January 2018

Mouse oocytes nucleoli rescue embryonic development of porcine enucleolated oocytes.

Zygote 2017 Dec 20;25(6):675-685. Epub 2017 Nov 20.

Constantine the Philosopher University,Nitra,Slovak Republic.

It is well known that nucleoli of fully grown mammalian oocytes are indispensable for embryonic development. Therefore, the embryos originated from previously enucleolated (ENL) oocytes undergo only one or two cleavages and then their development ceases. In our study the interspecies (mouse/pig) nucleolus transferred embryos (NuTE) were produced and their embryonic development was analyzed by autoradiography, transmission electron microscopy (TEM) and immunofluorescence (C23 and upstream binding factor (UBF)). Our results show that the re-injection of isolated oocyte nucleoli, either from the pig (P + P) or mouse (P + M), into previously enucleolated and subsequently matured porcine oocytes rescues their development after parthenogenetic activation and some of these develop up to the blastocyst stage (P + P, 11.8%; P + M, 13.5%). In nucleolus re-injected 8-cell and blastocyst stage embryos the number of nucleoli labeled with C23 in P + P and P + M groups was lower than in control (non-manipulated) group. UBF was localized in small foci within the nucleoli of blastocysts in control and P + P embryos, however, in P + M embryos the labeling was evenly distributed in the nucleoplasm. The TEM and autoradiographic evaluations showed the formation of functional nucleoli and de novo rRNA synthesis at the 8-cell stage in both, control and P + P group. In the P + M group the formation of comparable nucleoli was delayed. In conclusion, our results indicate that the mouse nucleolus can rescue embryonic development of enucleolated porcine oocytes, but the localization of selected nucleolar proteins, the timing of transcription activation and the formation of the functional nucleoli in NuTE compared with control group show evident aberrations.
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http://dx.doi.org/10.1017/S0967199417000491DOI Listing
December 2017

Intrinsically Defective Microtubule Dynamics Contribute to Age-Related Chromosome Segregation Errors in Mouse Oocyte Meiosis-I.

Curr Biol 2017 Apr;27(7):1040-1047

Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montreal, QC H2X 0A9, Canada; Department of Obstetrics and Gynaecology, University of Montréal, Montreal, QC H3T 1J4, Canada. Electronic address:

Chromosome segregation errors in mammalian oocytes compromise development and are particularly prevalent in older females, but the aging-related cellular changes that promote segregation errors remain unclear [1, 2]. Aging causes a loss of meiotic chromosome cohesion, which can explain premature disjunction of sister chromatids [3-7], but why intact sister pairs should missegregate in meiosis-I (termed non-disjunction) remains unknown. Here, we show that oocytes from naturally aged mice exhibit substantially altered spindle microtubule dynamics, resulting in transiently multipolar spindles that predispose the oocytes to kinetochore-microtubule attachment defects and missegregation of intact sister chromatid pairs. Using classical micromanipulation approaches, including reciprocally transferring nuclei between young and aged oocytes, we show that altered microtubule dynamics are not attributable to age-related chromatin changes. We therefore report that altered microtubule dynamics is a novel primary lesion contributing to age-related oocyte segregation errors. We propose that, whereas cohesion loss can explain premature sister separation, classical non-disjunction is instead explained by altered microtubule dynamics, leading to aberrant spindle assembly.
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http://dx.doi.org/10.1016/j.cub.2017.02.025DOI Listing
April 2017

Quantitative Microinjection of Morpholino Antisense Oligonucleotides into Mouse Oocytes to Examine Gene Function in Meiosis-I.

Methods Mol Biol 2016 ;1457:217-30

Centre de Recherche du Centre Hospitalier de l'Université de Montréal, 900 Rue St. Denis, Montreal, QC, Canada, H2X 0A9.

Specific protein depletion is a powerful approach for assessing individual gene function in cellular processes, and has been extensively employed in recent years in mammalian oocyte meiosis-I. Conditional knockout mice and RNA interference (RNAi) methods such as siRNA or dsRNA microinjection are among several approaches to have been applied in this system over the past decade. RNAi by microinjection of Morpholino antisense Oligonucleotides (MO), in particular, has proven highly popular and tractable in many studies, since MOs have high specificity of interaction, low cell toxicity, and are more stable than other microinjected RNAi molecules. Here, we describe a method of MO microinjection into the mouse germinal vesicle-stage (GV) oocyte followed by a simple immunofluorescence approach for examination of gene function in meiosis-I.
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http://dx.doi.org/10.1007/978-1-4939-3795-0_16DOI Listing
January 2018

Ca(2+) dynamics in oocytes from naturally-aged mice.

Sci Rep 2016 Jan 20;6:19357. Epub 2016 Jan 20.

Centre Recherche Centre Hospitalier Université de Montréal, Montreal, Québec, Canada, H2X 0A9.

The ability of human metaphase-II arrested eggs to activate following fertilisation declines with advancing maternal age. Egg activation is triggered by repetitive increases in intracellular Ca(2+) concentration ([Ca(2+)]i) in the ooplasm as a result of sperm-egg fusion. We therefore hypothesised that eggs from older females feature a reduced ability to mount appropriate Ca(2+) responses at fertilisation. To test this hypothesis we performed the first examination of Ca(2+) dynamics in eggs from young and naturally-aged mice. Strikingly, we find that Ca(2+) stores and resting [Ca(2+)]i are unchanged with age. Although eggs from aged mice feature a reduced ability to replenish intracellular Ca(2+) stores following depletion, this difference had no effect on the duration, number, or amplitude of Ca(2+) oscillations following intracytoplasmic sperm injection or expression of phospholipase C zeta. In contrast, we describe a substantial reduction in the frequency and duration of oscillations in aged eggs upon parthenogenetic activation with SrCl2. We conclude that the ability to mount and respond to an appropriate Ca(2+) signal at fertilisation is largely unchanged by advancing maternal age, but subtle changes in Ca(2+) handling occur that may have more substantial impacts upon commonly used means of parthenogenetic activation.
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http://dx.doi.org/10.1038/srep19357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726220PMC
January 2016

De novo DNA methylation drives 5hmC accumulation in mouse zygotes.

Nat Cell Biol 2016 Feb 11;18(2):225-233. Epub 2016 Jan 11.

Medical Research Council Clinical Sciences Centre, Imperial College London, London W12 0NN, United Kingdom.

Zygotic epigenetic reprogramming entails genome-wide DNA demethylation that is accompanied by Tet methylcytosine dioxygenase 3 (Tet3)-driven oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC; refs 1-4). Here we demonstrate using detailed immunofluorescence analysis and ultrasensitive LC-MS-based quantitative measurements that the initial loss of paternal 5mC does not require 5hmC formation. Small-molecule inhibition of Tet3 activity, as well as genetic ablation, impedes 5hmC accumulation in zygotes without affecting the early loss of paternal 5mC. Instead, 5hmC accumulation is dependent on the activity of zygotic Dnmt3a and Dnmt1, documenting a role for Tet3-driven hydroxylation in targeting de novo methylation activities present in the early embryo. Our data thus provide further insights into the dynamics of zygotic reprogramming, revealing an intricate interplay between DNA demethylation, de novo methylation and Tet3-driven hydroxylation.
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http://dx.doi.org/10.1038/ncb3296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765106PMC
February 2016

Mastl is required for timely activation of APC/C in meiosis I and Cdk1 reactivation in meiosis II.

J Cell Biol 2014 Sep 22;206(7):843-53. Epub 2014 Sep 22.

Department of Chemistry and Molecular Biology, University of Gothenburg, S-405 30 Gothenburg, Sweden

In mitosis, the Greatwall kinase (called microtubule-associated serine/threonine kinase like [Mastl] in mammals) is essential for prometaphase entry or progression by suppressing protein phosphatase 2A (PP2A) activity. PP2A suppression in turn leads to high levels of Cdk1 substrate phosphorylation. We have used a mouse model with an oocyte-specific deletion of Mastl to show that Mastl-null oocytes resume meiosis I and reach metaphase I normally but that the onset and completion of anaphase I are delayed. Moreover, after the completion of meiosis I, Mastl-null oocytes failed to enter meiosis II (MII) because they reassembled a nuclear structure containing decondensed chromatin. Our results show that Mastl is required for the timely activation of anaphase-promoting complex/cyclosome to allow meiosis I exit and for the rapid rise of Cdk1 activity that is needed for the entry into MII in mouse oocytes.
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http://dx.doi.org/10.1083/jcb.201406033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4178961PMC
September 2014

Vitrification of fully grown and growing porcine oocytes using germinal vesicle transfer.

J Reprod Dev 2011 Jun 28;57(3):335-41. Epub 2011 Jan 28.

Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Kasama, Japan.

The survival rate of vitrified germinal vesicle (GV) stage porcine oocytes is very low, and it is not known if the vitrification damages the nucleus, cytoplasm or both. We have evaluated the eventual GV or cytoplasmic damage in fully grown (FG) and growing vitrified oocytes. Fifty-five percent of nonvitrified FG cumulus-denuded oocytes reached the metaphase II (MII) stage in culture. When growing oocytes from preantral (PA) and early antral (EA) follicles were matured in vitro, almost all oocytes were arrested at the GV stage (GV stage: PA 88.9 and EA 79.5%, respectively). When fresh GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes and matured in vitro, some of them reached the MII stage (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). The maturation rate of vitrified FG oocytes was only 6.1% but increased dramatically when vitrified GVs from FG, PA and EA oocytes were transferred into fresh enucleated FG oocytes (MII stage: VitFG/FG 43.9%, VitPA/FG 7.1% and VitEA/FG 26.3%, respectively). These results were not significantly different from those for the nonvitrified groups (MII stage: FG/FG 57.5%, PA/FG 9.3% and EA/FG 35.3%, respectively). We activated the reconstructed oocytes that received fresh or vitrified GVs (FG/FG, EA/FG, VitFG/FG and VitEA/FG) and examined their embryonic development. Cleaved embryos (nonvitrified groups 13.0-61.8%, vitrified groups 33.3-40.0%) and blastocysts (nonvitrified groups 0.0-18.2%, vitrified groups 0.0-2.9%) were obtained after activation. These results demonstrate that vitrified porcine GVs maintain maturational and developmental competence and that vitrification predominantly damages the cytoplasm.
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http://dx.doi.org/10.1262/jrd.10-177hDOI Listing
June 2011

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor.

Cryobiology 2008 Dec 22;57(3):269-75. Epub 2008 Sep 22.

Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca(2+) chelator and mitochondrial Ca(2+) uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P<0.05) than those in the other vitrified groups (10.3-12.3% and 6.2-10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7-21.6% and 8.5-155%, respectively). When BAPTA-AM, an intracellular Ca(2+) chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5-36.2% and 25.0-26.9%, respectively) were significantly higher (P<0.05) than those in the absent vitrified groups (17.2-24.2% and 12.9-19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca(2+) uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8-38.4% and 14.4-24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization. These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.
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http://dx.doi.org/10.1016/j.cryobiol.2008.09.008DOI Listing
December 2008

Molecular cloning of a porcine (Sus scrofa) apoptosis inhibitory ligand, netrin-1, and its receptor, p53RDL1.

J Reprod Dev 2008 Aug 23;54(4):275-80. Epub 2008 May 23.

Animal Resource Science Center, The University of Tokyo, Kasama, Japan.

The apoptosis inhibitory ligand (Netrin-1) and its receptor (p53-regulated receptor for death and life: p53RDL1) play an important role in the regulation of selective apoptosis. When Netrin-1 binds to p53RDL1, p53-dependent apoptosis is inhibited. We identified porcine (Sus scrofa) cDNAs encoding Netrin-1 [pNetrin-1; 1,803 base pairs (bp) and 600 amino acids (aa)] and p53RDL1 (pp53RDL1; 2,838 bp and 945 aa). Porcine p53RDL1 (pp53RDL1) contains a death domain (DD), a tandem specific amino acid region, in its C-terminal, suggesting that it mediates death signaling by binding with other pro-apoptotic factors via the DD. Porcine Netrin-1 (pNetrin-1), pp53RDL1 and the DD in pp53RDL1 showed high levels of identity in aa sequence with human and murine Netrin-1 (98 and 97%, respectively), p53RDL1 (94 and 91%, respectively) and the DD in p53RDL1 (96 and 95%, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) revealed that the levels of pNetrin-1 and pp53RDL1 mRNAs were moderate in granulosa cells compared with their expression in other tissues and that their levels during follicular atresia were stable. The Netrin-1 and p53RDL1 system may regulate the induction of apoptosis in porcine granulosa cells.
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http://dx.doi.org/10.1262/jrd.20044DOI Listing
August 2008

Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase Czeta.

Reproduction 2006 Sep;132(3):393-401

Laboratory of Animal Breeding and Reproduction, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

The molecular mechanism by which sperm triggers Ca2+ oscillation, oocyte activation, and early embryonic development has not been clarified. Recently, oocyte activation has been shown to be induced by sperm-specific phospholipase Czeta (PLCzeta). The ability of PLCzeta to induce oocyte activation is highly conserved across vertebrates. In the present study, porcine PLCzeta cDNA was identified and the nucleotide sequence was determined. The expression pattern of porcine PLCzeta mRNA during the period of postnatal testicular development was shown to be similar to that of mouse PLCzeta. PLCzeta mRNA expression in the pig and mouse was detected only in the testes when the elongated spermatids had differentiated, and was detected from day 96 after birth in the pig. Histological examination of porcine testis during the period of postnatal development revealed the presence of spermatozoa from day 110 after birth. These findings suggest that the synthesis of PLCzeta mRNA starts when spermiogenesis is initiated. Microinjection of porcine PLCzeta complementary RNA into porcine oocytes demonstrated that porcine PLCzeta has the ability to trigger repetitive Ca2+ transients in porcine oocytes similar to that observed during fertilization. It was also found that porcine PLCzeta cRNA has the potential to induce oocyte activation and initiate embryonic development up to the blastocyst stage.
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http://dx.doi.org/10.1530/rep.1.01018DOI Listing
September 2006