Publications by authors named "Masaru Matsuda"

44 Publications

The complete mitochondrial genome sequences of Japanese earthworms and (Clitellata: Megascolecidae).

Mitochondrial DNA B Resour 2021 Mar 18;6(3):965-967. Epub 2021 Mar 18.

Center for Bioscience Research and Education, Utsunomiya University, Utsunomiya, Japan.

Many studies have reported the complete mitochondrial genome sequences of Chinese Megascolecidae earthworms, however, there have been no reports on sequences originating from Japanese Megascolecidae earthworms. In this study, we determined complete mitochondrial genome sequences of two Japanese earthworms belonging to the complex within the Megascolecidae family. is one of the most common earthworms in Japan and was found to be morphologically similar to . The complete mitochondrial genomes of (15,186 bp; LC573968) and (15,109 bp; LC573969) contained typical 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic analysis revealed that these two species were sister species. Therefore, our findings will further contribute to phylogenetic and genetic diversity analyses of Megascolecidae earthworms.
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http://dx.doi.org/10.1080/23802359.2020.1830728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995826PMC
March 2021

Minimizing scattering-induced phase errors in differential interference contrast microscopy.

J Biomed Opt 2020 12;25(12)

Utsunomiya University, Department of Optical Engineering, Tochigi, Japan.

Significance: Differential interference contrast (DIC) microscopes allow noninvasive in vivo observation of transparent microstructures in tissue without the use of fluorescent dyes or genetic modification. We show how to modify a DIC microscope to measure the sample phase distribution accurately and in real-time even deep inside sample tissue.

Aim: Our aim is to improve the DIC microscope's phase measurement to remove the phase bias that occurs in the presence of strong scattering.

Approach: A quarter-wave plate was added in front of the polarization camera, allowing a modified phase calculation to incorporate all four polarization orientation angles (0 deg, 45 deg, 90 deg, and 135 deg) captured simultaneously by the polarization camera, followed by deconvolution.

Results: We confirm that the proposed method reduces phase measurement error in the presence of scattering and demonstrate the method using in vivo imaging of a beating heart inside a medaka egg and the whole-body blood circulation in a young medaka fish.

Conclusions: Modifying a polarization-camera DIC microscope with a quarter-wave plate allows users to image deep inside samples without phase bias due to scattering effects.
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http://dx.doi.org/10.1117/1.JBO.25.12.123703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7734411PMC
December 2020

Parallel Evolution of Two dmrt1-Derived Genes, dmy and dm-W, for Vertebrate Sex Determination.

iScience 2020 Jan 4;23(1):100757. Epub 2019 Dec 4.

Department of Bioscience, School of Science, Kitasato University, 1-15-1 Kitasato, Minamiku, Sagamihara, Kanagawa 252-0373, Japan. Electronic address:

Animal sex-determining genes, which bifurcate for female and male development, are diversified even among closely related species. Most of these genes emerged independently from various sex-related genes during species diversity as neofunctionalization-type genes. However, the common mechanisms of this divergent evolution remain poorly understood. Here, we compared the molecular evolution of two sex-determining genes, the medaka dmy and the clawed frog dm-W, which independently evolved from the duplication of the transcription factor-encoding masculinization gene dmrt1. Interestingly, we detected parallel amino acid substitutions, from serine (S) to threonine (T), on the DNA-binding domains of both ancestral DMY and DM-W, resulting from positive selection. Two types of DNA-protein binding experiments and a luciferase reporter assay demonstrated that these S-T substitutions could strengthen the DNA-binding abilities and enhance the transcriptional regulation function. These findings suggest that the parallel S-T substitutions may have contributed to the establishment of dmy and dm-W as sex-determining genes.
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http://dx.doi.org/10.1016/j.isci.2019.100757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941857PMC
January 2020

Erratum: Video-rate quantitative phase analysis by a DIC microscope using a polarization camera: errata.

Biomed Opt Express 2019 06 23;10(6):2967-2968. Epub 2019 May 23.

Department of Optical Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi, Japan.

[This corrects the article on p. 1273 in vol. 10, PMID: 30891345.].
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http://dx.doi.org/10.1364/BOE.10.002967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6583344PMC
June 2019

Video-rate quantitative phase analysis by a DIC microscope using a polarization camera.

Biomed Opt Express 2019 Mar 19;10(3):1273-1281. Epub 2019 Feb 19.

Department of Optical Engineering, Utsunomiya University, 7-1-2 Yoto, Utsunomiya, Tochigi, Japan.

This paper describes how to take advantage of the replacement of an intensity camera with a polarization camera in a standard differential interference contrast (DIC) microscope. Using a polarization camera enables snapshot quantitative phase analysis so that real-time imaging of living transparent tissues become possible. Using our method, we quantify the phase measurement accuracy using a phantom consisting of glass beads embedded in lacquer. In order to demonstrate these advantages, we image the pumping heart and blood flow in a living egg.
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http://dx.doi.org/10.1364/BOE.10.001273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420286PMC
March 2019

Structural investigation of α-l-fucosidase from the pancreas of Patiria pectinifera, based on molecular cloning.

Carbohydr Res 2019 Mar 7;475:27-33. Epub 2019 Feb 7.

Department of Applied Chemistry and Biochemistry, National Institute of Technology, Fukushima College, 30 Nagao, Iwaki, Fukushima, 970-8034, Japan. Electronic address:

An α-l-fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6 kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70 °C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galβ1→4Glc-β-pNP). We determined the primary structure of the α-l-fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α-l-fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.
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http://dx.doi.org/10.1016/j.carres.2019.02.001DOI Listing
March 2019

Evolution of the sex-determining gene in the teleostean genus Oryzias.

Gen Comp Endocrinol 2016 12 9;239:80-88. Epub 2015 Oct 9.

Department of Environmental Sciences, Faculty of Science, Niigata University, Niigata 950-2181, Japan. Electronic address:

In the genetic sex determination of vertebrates, the gonadal sex depends on the combination of sex chromosomes that a zygote possesses. Despite the discovery of the sex-determining gene (SRY/Sry) in mammals in 1990s, the sex-determining gene in non-mammalian vertebrates remained an enigma for over a decade. In most mammals, the male-inducing master sex-determining gene is located on the Y chromosome and is therefore absent from XX females. A second sex-determining gene, Dmy, was described in the Oryzias latipes in 2002 and has a DNA-binding motif that is different from the motif in the mammalian sex-determining gene SRY or Sry. Dmy is also located on the Y chromosome and is therefore absent in XX females. Seven other sex-determining genes, including candidate genes, are now known in birds, a frog species, and 5 fish species. These findings over the past twenty years have increased our knowledge of sex-determining genes and sex chromosomes among vertebrates. Here, we review recent advances in our understanding of sex-determining genes and genetic sex determination systems in fish, especially those of the Oryzias species, which are described in detail. The facts suggest some patterns of how new sex-determining genes emerged and evolved. We believe that these facts are common not only in Oryzias but also in other fish species. This knowledge will help to elucidate the conserved mechanisms from which various sex-determining mechanisms have evolved.
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http://dx.doi.org/10.1016/j.ygcen.2015.10.004DOI Listing
December 2016

Mutation of Gonadal soma-derived factor induces medaka XY gonads to undergo ovarian development.

Biochem Biophys Res Commun 2015 Nov 25;467(1):109-14. Epub 2015 Sep 25.

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan; Center for Bioscience Research and Education, Utsunomiya University, Tochigi 321-8505, Japan. Electronic address:

Gonochoristic species have a bipotential gonad that develops into a testis or an ovary. In species whose sex is determined by a genetic factor, the expression of a sex-determining gene is the first cue that directs the development of a bipotential gonad. Subsequent expression of downstream genes induces the gonad to develop into a testis or an ovary. The TGF-ß family member Gonadal soma-derived factor (Gsdf) is thought to be an important gene for gonadal development in teleost fish, and it is expressed at higher levels in the testis than in the ovary from early to mature stages. However, there is little functional information about the gene. In this study, we targeted the Gsdf coding region in the medaka fish Oryzias latipes using transcription activator-like effector nucleases (TALENs) and studied the phenotypes of the Gsdf mutant medaka. Although normal and heterozygous XY gonads developed into a testis, all XY gonads with a homozygous mutation in Gsdf developed into an ovary at early developmental stages. However, two-thirds of Gsdf mutant XY gonads developed into testes in the adult stages. These results demonstrate that although a gonad can develop into a complete testis in the absence of Gsdf, Gsdf function is critical for directing the bipotential gonad at early developmental stages. Therefore, Gsdf is an endogenous inducer of testicular development similar to a master sex-determining gene.
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http://dx.doi.org/10.1016/j.bbrc.2015.09.112DOI Listing
November 2015

Developmental and morphological studies in Japanese medaka with ultra-high resolution optical coherence tomography.

Biomed Opt Express 2015 Feb 6;6(2):297-308. Epub 2015 Jan 6.

Center for Optical Research and Education (CORE), Utsunomiya University, Japan.

We propose ultra-high resolution optical coherence tomography to study the morphological development of internal organs in medaka fish in the post-embryonic stages at micrometer resolution. Different stages of Japanese medaka were imaged after hatching in vivo with an axial resolution of 2.8 µm in tissue. Various morphological structures and organs identified in the OCT images were then compared with the histology. Due to the medaka's close resemblance to vertebrates, including humans, these morphological features play an important role in morphogenesis and can be used to study diseases that also occur in humans.
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http://dx.doi.org/10.1364/BOE.6.000297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354602PMC
February 2015

The habenulo-raphe serotonergic circuit encodes an aversive expectation value essential for adaptive active avoidance of danger.

Neuron 2014 Dec 20;84(5):1034-48. Epub 2014 Nov 20.

Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute, Saitama 351-0198, Japan; Laboratory for Molecular Brain Science, Department of Life Science and Medical Bioscience, Waseda University, Tokyo 162-8430, Japan; Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan. Electronic address:

Anticipation of danger at first elicits panic in animals, but later it helps them to avoid the real threat adaptively. In zebrafish, as fish experience more and more danger, neurons in the ventral habenula (vHb) showed tonic increase in the activity to the presented cue and activated serotonergic neurons in the median raphe (MR). This neuronal activity could represent the expectation of a dangerous outcome and be used for comparison with a real outcome when the fish is learning how to escape from a dangerous to a safer environment. Indeed, inhibiting synaptic transmission from vHb to MR impaired adaptive avoidance learning, while panic behavior induced by classical fear conditioning remained intact. Furthermore, artificially triggering this negative outcome expectation signal by optogenetic stimulation of vHb neurons evoked place avoidance behavior. Thus, vHb-MR circuit is essential for representing the level of expected danger and behavioral programming to adaptively avoid potential hazard.
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http://dx.doi.org/10.1016/j.neuron.2014.10.035DOI Listing
December 2014

TALEN-mediated mutagenesis in zebrafish reveals a role for r-spondin 2 in fin ray and vertebral development.

FEBS Lett 2014 Dec 1;588(24):4543-50. Epub 2014 Nov 1.

Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. Electronic address:

R-spondin (Rspo) encodes a multi-domain protein that modulates the Wnt-signaling pathway. Two distinct rspo2 zebrafish mutants were generated by TALEN-mediated mutagenesis: a null mutant, rspo2(null), lacking all functional domains, and a hypomorphic mutant, rspo2(tsp), lacking the two N-terminal domains. Mutants were analyzed mainly for abnormalities in the skeletal system. Fin ray skeletons were formed normally in the rspo2(tsp) mutants, but were absent from the rspo2(null) mutants. Hypoplasia of the neural/hemal arches and ribs was observed in both mutants. Thus, the two rspo2 mutants help to identify the functions of Rspo2 in skeletogenesis, as well as functional differences among multiple Rspo2 domains.
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http://dx.doi.org/10.1016/j.febslet.2014.10.015DOI Listing
December 2014

Co-option of Sox3 as the male-determining factor on the Y chromosome in the fish Oryzias dancena.

Nat Commun 2014 Jun 20;5:4157. Epub 2014 Jun 20.

1] Laboratory of Bioresources, National Institute for Basic Biology, Okazaki 444-8585, Japan [2] Department of Basic Biology, the Graduate University for Advanced Studies (SOKENDAI), Okazaki 444-8585, Japan.

Sex chromosomes harbour a primary sex-determining signal that triggers sexual development of the organism. However, diverse sex chromosome systems have been evolved in vertebrates. Here we use positional cloning to identify the sex-determining locus of a medaka-related fish, Oryzias dancena, and find that the locus on the Y chromosome contains a cis-regulatory element that upregulates neighbouring Sox3 expression in developing gonad. Sex-reversed phenotypes in Sox3(Y) transgenic fish, and Sox3(Y) loss-of-function mutants all point to its critical role in sex determination. Furthermore, we demonstrate that Sox3 initiates testicular differentiation by upregulating expression of downstream Gsdf, which is highly conserved in fish sex differentiation pathways. Our results not only provide strong evidence for the independent recruitment of Sox3 to male determination in distantly related vertebrates, but also provide direct evidence that a novel sex determination pathway has evolved through co-option of a transcriptional regulator potentially interacted with a conserved downstream component.
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http://dx.doi.org/10.1038/ncomms5157DOI Listing
June 2014

Spontaneous germline excision of Tol1, a DNA-based transposable element naturally occurring in the medaka fish genome.

Genome 2014 Apr 12;57(4):193-9. Epub 2014 May 12.

a Matsuyama High School, Higashimatsuyama 355-0018, Japan.

DNA-based transposable elements are ubiquitous constituents of eukaryotic genomes. Vertebrates are, however, exceptional in that most of their DNA-based elements appear to be inactivated. The Tol1 element of the medaka fish, Oryzias latipes, is one of the few elements for which copies containing an undamaged gene have been found. Spontaneous transposition of this element in somatic cells has previously been demonstrated, but there is only indirect evidence for its germline transposition. Here, we show direct evidence of spontaneous excision in the germline. Tyrosinase is the key enzyme in melanin biosynthesis. In an albino laboratory strain of medaka fish, which is homozygous for a mutant tyrosinase gene in which a Tol1 copy is inserted, we identified de novo reversion mutations related to melanin pigmentation. The gamete-based reversion rate was as high as 0.4%. The revertant fish carried the tyrosinase gene from which the Tol1 copy had been excised. We previously reported the germline transposition of Tol2, another DNA-based element that is thought to be a recent invader of the medaka fish genome. Tol1 is an ancient resident of the genome. Our results indicate that even an old element can contribute to genetic variation in the host genome as a natural mutator.
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http://dx.doi.org/10.1139/gen-2014-0011DOI Listing
April 2014

Ol4E-T, a eukaryotic translation initiation factor 4E-binding protein of medaka fish (Oryzias latipes), can interact with nanos3 and vasa in vitro.

J Exp Zool B Mol Dev Evol 2013 Jan 5;320(1):10-21. Epub 2012 Sep 5.

Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan, China.

Maternal factors have essential roles in the specification and development of germ cells in metazoans. In Drosophila, a number of genes such as oskar, vasa, nanos, and tudor are required for specific steps in pole cell formation and further germline development. Drosophila cup, another maternal factor, is confirmed as a main factor in normal oogenesis, maintenance, and survival of female germ-line stem cells by interaction with Nanos. Through searching for the homolog of Drosophila cup in the medaka, the homolog of eukaryotic translation initiation factor 4E (eIF4E)-transporter, named Ol4E-T, was identified. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that Ol4E-T is maternally deposited in the embryo and Ol4E-T expression is maintained throughout embryogenesis. Ol4E-T is predominantly expressed in the adult gonads. In the testes, Ol4E-T is expressed in the same regions where medaka vasa, named olvas is expressed. In the ovary, expression of Ol4E-T conforms to that of nanos3 and olvas. Ol4E-T harbors a well-conserved eIF4E-binding motif, YTKEELL, by which Ol4E-T interacts with eIF4E in medaka. Additionally, Ol4E-T can interact with medaka Nanos3 and Olvas, as shown by yeast two hybridization. The spatial expression and interactions between Ol4E-T with germ cell markers Olvas and Nanos3 suggest a role for Ol4E-T in germ-line development in medaka.
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http://dx.doi.org/10.1002/jez.b.22465DOI Listing
January 2013

Tracing the emergence of a novel sex-determining gene in medaka, Oryzias luzonensis.

Genetics 2012 May 23;191(1):163-70. Epub 2012 Feb 23.

Institute of Science and Technology, Niigata University, Niigata 950-2181, Japan.

Three sex-determining (SD) genes, SRY (mammals), Dmy (medaka), and DM-W (Xenopus laevis), have been identified to date in vertebrates. However, how and why a new sex-determining gene appears remains unknown, as do the switching mechanisms of the master sex-determining gene. Here, we used positional cloning to search for the sex-determining gene in Oryzias luzonensis and found that GsdfY (gonadal soma derived growth factor on the Y chromosome) has replaced Dmy as the master sex-determining gene in this species. We found that GsdfY showed high expression specifically in males during sex differentiation. Furthermore, the presence of a genomic fragment that included GsdfY converts XX individuals into fertile XX males. Luciferase assays demonstrated that the upstream sequence of GsdfY contributes to the male-specific high expression. Gsdf is downstream of Dmy in the sex-determining cascade of O. latipes, suggesting that emergence of the Dmy-independent Gsdf allele led to the appearance of this novel sex-determining gene in O. luzonensis.
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http://dx.doi.org/10.1534/genetics.111.137497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338257PMC
May 2012

Expression of 3β-hydroxysteroid dehydrogenase (hsd3b), star and ad4bp/sf-1 during gonadal development in medaka (Oryzias latipes).

Gen Comp Endocrinol 2012 Apr 6;176(2):222-30. Epub 2012 Feb 6.

Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano 390-8621, Japan.

In most vertebrates, sex steroids play a critical role in gonadal development, maturation of germ cells, and development of secondary sexual characteristics. Sex steroids are synthesized in steroid-producing cells (SPCs) in the testis known as Leydig cells, as well as in thecal and granulosa cells in the ovary. In SPCs, cholesterol is sequentially catalyzed by a set of steroidogenic factors and enzymes in order to produce sex steroids. Therefore, integrated expression of the genes involved in steroidogenesis is critical for the proper production of sex steroids. In the present study, regulatory mechanisms of steroidogenic factors and enzymes were examined. We focused on hsd3b, star and ad4bp/sf-1 as well as the description of temporal and spatial expression of these genes during gonadal development in medaka (Oryzias latipes). During testicular development, hsd3b, star and ad4bp/sf-1 were co-expressed in the interstitial somatic cells subsequent to the formation of the seminiferous tubule precursor, suggesting that ad4bp/sf-1 regulated the transcription of both hsd3b and star. During ovarian development, the expression pattern of hsd3b coincided with that of cyp11a1, but not with that of aromatase. Although ad4bp/sf-1 was mainly expressed in presumptive follicular cells, it was also detected in hsd3b positive interstitial cells in the developing ovary. Contrary to our expectations, the onset of star expression occurred during a later stage of ovarian development than the expression of other steroidogenic enzymes. Thus, the regulation mechanism of star transcription appears to differ from that of the other steroidogenic enzymes in the developing ovary, but not in the developing testis.
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http://dx.doi.org/10.1016/j.ygcen.2012.01.019DOI Listing
April 2012

Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka.

Chromosome Res 2012 Jan;20(1):163-76

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.

DMRT1, which is found in many vertebrates, exhibits testis-specific expression during the sexual differentiation period, suggesting a conserved function of DMRT1 in the testicular development of vertebrate gonads. However, functional analyses have been reported only in mammals. The current study focused on the Dmrt1 function in the teleost medaka, Oryzias latipes, which has an XX-XY sex determination system. Although medaka sex is determined by the presence or absence of the Y chromosome-specific gene Dmy, we demonstrated that in one Dmrt1 mutant line, which was found by screening a gene-driven mutagenesis library, XY mutants developed into normal females and laid eggs. Histological analyses of this mutant revealed that the XY mutant gonads first developed into the normal testis type. However, the gonads transdifferentiated into the ovary type. The mutant phenotype could be rescued by transgenesis of the Dmrt1 genomic region. These results show that Dmrt1 is essential to maintain testis differentiation after Dmy-triggered male differentiation pathway.
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http://dx.doi.org/10.1007/s10577-011-9264-xDOI Listing
January 2012

Expression of gonadal soma derived factor (GSDF) is spatially and temporally correlated with early testicular differentiation in medaka.

Gene Expr Patterns 2010 Sep 17;10(6):283-9. Epub 2010 Jun 17.

Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

In the teleost fish, medaka (Oryzias latipes), the sex is genetically determined at the time of fertilization. The males are heterogametic with XY chromosome composition, while females are of XX chromosome composition. The male sexual differentiation is initiated in XY embryos of medaka by the sex-determining gene Dmy. In this study, we have cloned the gonadal soma derived factor (Gsdf) from medaka and characterized its expression pattern during the initiation of morphological testicular differentiation. By real-time PCR, an XY-specific up-regulation was detected in the expression levels of Gsdf in the whole embryos of medaka at 6days post fertilization (dpf), coincident with the initiation of testicular differentiation in the XY gonads. Whole mount and section in situ hybridizations reaffirmed that Gsdf was expressed exclusively in primordial gonads of only the genetic males at 6dpf. Conversely, the expression of Gsdf was found to be very weak in the XX gonads during embryogenesis. Importantly, Gsdf and Dmy were found to be co-localized in the same somatic cells in the XY gonads. When the XY embryos were treated with estradiol-17beta, in order to reverse their phenotypic sex, a decline was observed in the expression of Gsdf in these embryos by real-time PCR.
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http://dx.doi.org/10.1016/j.gep.2010.06.005DOI Listing
September 2010

AAB-sequence living radical chain copolymerization of naturally occurring limonene with maleimide: an end-to-end sequence-regulated copolymer.

J Am Chem Soc 2010 Jul;132(29):10003-5

Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan.

Sequence control in chain-growth polymerization is still one of the most challenging topics in synthetic polymer chemistry in contrast to natural macromolecules with completely sequence-regulated structures like proteins and DNA. Here, we report the quantitative and highly selective 1:2 sequence-regulated radical copolymerization of naturally occurring (+)-d-limonene (L) and a maleimide (M) in fluoroalcohol giving chiral copolymers with high glass transition temperatures (220-250 degrees C) originating from the specific rigid cyclic structures of the monomers. Furthermore, the combination with a reversible addition-fragmentation chain transfer (RAFT) agent (C-S) via the controlled/living radical polymerization resulted in end-to-end sequence-regulated copolymers [C-(M-M-L)(n)-M-S] with both highly sequenced chain ends and main-chain repeating units as well as controlled molecular weights.
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http://dx.doi.org/10.1021/ja1042353DOI Listing
July 2010

Cloning and expression of medaka cholesterol side chain cleavage cytochrome P450 during gonadal development.

Dev Growth Differ 2010 May;52(4):385-95

Department of Biology, Faculty of Science, Shinshu University, Matsumoto, Nagano, Japan.

Cholesterol side chain cleavage cytochrome P450 (P450scc, Cyp11a) is responsible for the first step in steroidogenesis, catalyzing the conversion of cholesterol to prognenolone. To investigate the differentiation of steroid-producing cells and the function of sex steroids during gonadal differentiation in the teleost fish, medaka (Oryzias latipes), we isolated the full length cDNA of medaka P450scc and analyzed the expression pattern of P450scc mRNA during gonadal development using in situ hybridization. At hatching, and just after the initiation of morphological sex differentiation, we did not detect any P450scc expression in both sexes. In male gonads, expression of P450scc was detected in the interstitial somatic cells 15 days after hatching following the formation of the seminiferous tubule precursor, and was maintained in the interstitial somatic cells throughout testicular development. In the female gonad, expression of P450scc was initially detected in interstitial somatic cells 5 days after hatching. Subsequently, the expression of P450scc was continuously detected in the interstitial somatic cells of the developing ovary. This expression pattern of P450scc differed from that of female specific steroidogenic enzyme P450arom. Both P450scc and P450arom expressing cells, only P450scc expressing cells, and only P450arom expressing cells were observed. Our results suggest that expression of steroidogenic enzymes is regulated by various mechanisms during ovarian development.
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http://dx.doi.org/10.1111/j.1440-169X.2010.01178.xDOI Listing
May 2010

Doublesex- and Mab-3-related transcription factor-1 repression of aromatase transcription, a possible mechanism favoring the male pathway in tilapia.

Endocrinology 2010 Mar 7;151(3):1331-40. Epub 2010 Jan 7.

Key Laboratory of Freshwater Fish Reproduction and Development (Ministry of Education), School of Life Science, Southwest University, 400715 Chongqing, People's Republic of China.

Doublesex- and Mab-3-related transcription factor-1 (Dmrt1) is an important transcription factor implicated in early testicular differentiation in vertebrates, but its target genes are largely unknown. In the Nile tilapia, estrogen is the natural inducer of ovarian differentiation. Our recent studies have shown that Forkhead-l2 up-regulated transcription of the Cyp19a1a gene (aromatase) in the gonads in a female-specific manner. However, the upstream factor(s) down-regulating Cyp19a1a expression during testicular differentiation remains unclear. In the present study, we used in vitro (promoter analysis) and in vivo (transgenesis and in situ hybridization) approaches to examine whether Dmrt1 inhibits Cyp19a1a's transcriptional activity. The in vitro analysis using luciferase assays revealed that Dmrt1 repressed basal as well as Ad4BP/SF-1-activated Cyp19a1a transcription in HEK 293 cells. Luciferase assays with various deletions of Dmrt1 also showed that the Doublesex and Mab-3 domain is essential for the repression. In vitro-translated Dmrt1 and the nuclear extract from tilapia testis could directly bind to the palindrome sequence ACATATGT in the Cyp19a1a promoter, as determined by EMSAs. Transgenic overexpression of Dmrt1 in XX fish resulted in decreased aromatase gene expression, reduced serum estradiol-17beta levels, retardation of the ovarian cavity's development, varying degrees of follicular degeneration, and even a partial to complete sex reversal. Our results indicate that aromatase is one of the targets of Dmrt1. Dmrt1 suppresses the female pathway by repressing aromatase gene transcription and estrogen production in the gonads of tilapia and possibly other vertebrates.
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http://dx.doi.org/10.1210/en.2009-0999DOI Listing
March 2010

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis.

Genesis 2009 May;47(5):289-99

Department of Biology, Shinshu University, Matsumoto, Nagano, Japan.

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX-XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species.
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http://dx.doi.org/10.1002/dvg.20498DOI Listing
May 2009

[Sex determination in the teleost medaka, Oryzias latipes].

Authors:
Masaru Matsuda

Tanpakushitsu Kakusan Koso 2008 Jul;53(9):1158-65

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July 2008

Stability in aromatase immunoreactivity of steroid-producing cells during early development of XX gonads of the Nile tilapia, Oreochromis niloticus: an organ culture study.

Zoolog Sci 2008 Mar;25(3):344-8

Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki, Aichi, Japan.

The organ culture system is a useful tool to study the effects of various factors on the development of undifferentiated gonads. In this study, we first established an organ culture system for gonads of all genetic male and female Nile tilapia at 5-122 days after hatching (dah). This short-term (3 days) organ culture system was then used to examine the stability of the immunoreactivity of aromatase (the enzyme which converts androgen to estrogen, thus playing a crucial role in ovarian differentiation) in steroid-producing cells (SPCs). Immunohistochemical analyses revealed that aromatase-positive cells could be initially detected in the vicinity of blood vessels in the XX gonads at 7 dah. These SPCs completely lost their immunoreactivity after 3 days in culture, indicating the instability of SPCs during early ovarian differentiation. In contrast, the immunoreactivity of the SPCs was maintained to some extent even after 3 days in culture, if the gonads were from 15-23 dah. In XX gonads collected at 122 dah, there were two major populations of SPCs: one in the vicinity of the blood vessel and the other near the oocyte. The aromatase immunoreactivity was maintained in SPCs located around the oocytes, but not in those in the vicinity of the blood vessel, after 3 days in culture. These results suggest that the SPCs originate from the cells in the vicinity of the blood vessels prior to the initial ovarian differentiation in tilapia and that the degree of differentiation of SPCs is dependent on their location in the ovary.
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http://dx.doi.org/10.2108/zsj.25.344DOI Listing
March 2008

Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a teleost fish, the medaka, Oryzias latipes.

Mol Reprod Dev 2007 Sep;74(9):1065-71

Department of Basic Biology, The Graduate University for Advanced Studies, Okazaki, Japan.

The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.
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http://dx.doi.org/10.1002/mrd.20497DOI Listing
September 2007

DMY gene induces male development in genetically female (XX) medaka fish.

Proc Natl Acad Sci U S A 2007 Mar 28;104(10):3865-70. Epub 2007 Feb 28.

Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates.
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http://dx.doi.org/10.1073/pnas.0611707104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1820675PMC
March 2007

Dax1 suppresses P450arom expression in medaka ovarian follicles.

Mol Reprod Dev 2007 Oct;74(10):1239-46

Department of Environmental System Science, Graduate School of Science and Technology, Shinshu University, Matsumoto, Japan.

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.
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http://dx.doi.org/10.1002/mrd.20689DOI Listing
October 2007

Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes.

Biochem Biophys Res Commun 2006 Dec 30;351(4):815-9. Epub 2006 Oct 30.

Laboratory of Reproductive Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

DMY is the second vertebrate sex-determining gene identified from the fish, Oryzias latipes. In this study, we used two different ways of sex reversal, DMY knock-down and estradiol-17beta (E2) treatment, to determine the possible function of DMY during early gonadal sex differentiation in XY medaka. Our findings revealed that the mitotic and meiotic activities of the germ cells in the 0 day after hatching (dah) DMY knock-down XY larvae were identical to those of the normal XX larvae, suggesting the microenvironment of these XY gonads to be similar to that of the normal XX gonad, where DMY is naturally absent. Conversely, E2 treatment failed to initiate mitosis in the XY gonad, possibly due to an active DMY, even though it could initiate meiosis. Present study is the first to prove that the germ cells in the XY gonad can resume the mitotic activity, if DMY was knocked down.
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http://dx.doi.org/10.1016/j.bbrc.2006.10.095DOI Listing
December 2006

Wild-derived XY sex-reversal mutants in the Medaka, Oryzias latipes.

Genetics 2006 Aug 15;173(4):2083-90. Epub 2006 May 15.

Graduate School of Science and Technology, Department of Environmental Science, Faculty of Science, Niigata University, Japan.

The medaka, Oryzias latipes, has an XX/XY sex-determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a sex-determining gene in this species. Previously, we found 23 XY sex-reversed females from 11 localities by examining the genotypic sex of wild-caught medaka. Genetic analyses revealed that all these females had Y-linked gene mutations. Here, we aimed to clarify the cause of this sex reversal. To achieve this, we screened for mutations in the amino acid coding sequence of DMY and examined DMY expression at 0 days after hatching (dah) using densitometric semiquantitative RT-PCR. We found that the mutants could be classified into two groups. One contained mutations in the amino acid coding sequence of DMY, while the other had reduced DMY expression at 0 dah although the DMY coding sequence was normal. For the latter, histological analyses indicated that YwOurYwOur (YwOur, Y chromosome derived from an Oura XY female) individuals with the lowest DMY expression among the tested mutants were expected to develop into females at 0 dah. These results suggest that early testis development requires DMY expression above a threshold level. Mutants with reduced DMY expression may prove valuable for identifying DMY regulatory elements.
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http://dx.doi.org/10.1534/genetics.106.058941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1569717PMC
August 2006

Molecular cloning and analysis of gonadal expression of Foxl2 in the medaka, Oryzias latipes.

Biochem Biophys Res Commun 2006 May;344(1):353-61

Department of Environmental System Science, Graduate School of Science and Technology, Shinshu University, Matsumoto 390-8621, Japan.

Foxl2 is a member of the winged helix/forkhead family of transcription factors and is known to be involved in ovarian development in some vertebrates. To address the role of Foxl2 in ovarian differentiation in medaka, we isolated Foxl2 cDNA and analyzed its expression patterns during sex differentiation. Expression of Foxl2 started in somatic cells surrounding germ cells in XX gonads, just after initiation of ovarian differentiation, and was maintained in granulosa cells throughout ovarian development. In the adult ovary, Foxl2 was expressed in previtellogenic and vitellogenic follicles, but expression ceased in postvitellogenic follicles. In contrast, Foxl2 mRNA could not be detected in testes. In addition, Foxl2 and aromatase mRNAs were co-localized in some somatic cells located on the ventral side of developing XX gonads. Our results suggested that Foxl2 was not involved in ovarian determination, but was involved in differentiation of granulosa cells in medaka.
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http://dx.doi.org/10.1016/j.bbrc.2006.03.137DOI Listing
May 2006