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論文

論文
Wakayama, Tomohiko ; Sai, Yoshimichi ; Ito, Akihiko ; Kato, Yukio ; Kurobo, Miho ; Murakami, Yoshinori ; Nakashima, Emi ; Tsuji, Akira ; Kitamura, Yukihiko ; Iseki, Shoichi ; 若山, 友彦 ; 崔, 吉道 ; 加藤, 将夫 ; 辻, 彰 ; 井関, 尚一
出版情報: Biology of Reproduction.  76  pp.1081-1090,  2007-06-01.  Society for the Study of Reproduction
URL: http://hdl.handle.net/2297/00049831
概要: 金沢大学医薬保健研究域医学系<br />The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spe rmatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis. © 2007 by the Society for the Study of Reproduction, Inc.<br />Embargo Period 12 months 続きを見る
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論文

論文
Sugimoto, Kazuhiro ; Koh, Eitetsu ; Sin, Ho-Su ; Maeda, Yuji ; Narimoto, Kazutaka ; Izumi, Koji ; Kobori, Yoshitomo ; Kitamura, Eiko ; Nagase, Hiroki ; Yoshida, Atsumi ; Namiki, Mikio
出版情報: Journal of Human Genetics.  54  pp.450-456,  2009-08-01.  Japan Society of Human Genetics = 日本人類遺伝学会 / Springer-Verlag Tokyo
URL: http://hdl.handle.net/2297/19417
概要: Numerous CpG islands containing tissue-specific differentially methylated regions (TDMRs) are potential methylation site s in normal cells and tissues. The VASA (also known as DDX4) gene is believed to be under the control of TDMRs. A total of 131 male patients with idiopathic azoospermia or severe oligospermia were evaluated histologically, and the methylation status of CpG islands in the VASA gene was screened. Genome DNAs were obtained from testicular biopsy and modified with sodium bisulfite, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied. This system is capable of analyzing both the methylated and unmethylated CpG island in the genome. The methylation analysis is conducted by an epigram as graphic data. On histological assessment, 17 of 131 patients revealed maturation arrest (MA).In all, 6 of the 17 patients showed particularly high VASA TDMR methylation rates, whereas the remaining 11 patients and controls had low methylation rates. This study may imply that the VASA TDMR methylation is significantly higher among patients with MA, in whom the VASA gene expression was silenced. This finding represents an important contribution to the molecular basis of meiotic arrest as one possible cause of idiopathic infertility. © 2009 The Japan Society of Human Genetics All rights reserved. 続きを見る
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論文

論文
仲田, 浩規 ; 若山, 友彦 ; 西内, 巧 ; 井関, 尚一
出版情報: Acta Histochemica et Cytochemica.  45  pp.47-56,  2012-01-01.  Japan Society of Histochemistry and Cytochemistry = 日本組織細胞化学会
URL: http://hdl.handle.net/2297/30293
概要: The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expres sed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells. © 2012 The Japan Society of Histochemistry and Cytochemistry.<br />金沢大学大学院医学系研究科 仲田 浩規 学位論文 / Thesis of Hiroki Nakata 続きを見る
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論文

論文
高, 栄哲 ; 飯島, 将司 ; 並木, 幹夫
出版情報: Journal of mammalian ova research = 日本卵子学会誌.  30  pp.135-144,  2013-12-05.  Japanese Society of Ova Research = 日本卵子学会
URL: http://hdl.handle.net/2297/40509
概要: 無精子症は精子形成に関与する多種多様な遺伝子群の異常で惹起されるが,精子形成責任遺伝子は未だ同定されていない.近年,遺伝子変異動物の作成による精子形成障害を示す例が少なからず報告され,精子形成に関与する遺伝子が多岐にわたることが明らかにされ ている.本稿では,遺伝子の視点から,減数分裂を特徴とする精子形成過程に深く関与する遺伝子群を概説し,無精子症を呈している疾患群から,その原因遺伝子について鳥瞰する.Y染色体長腕上にはAZF(Azoospermia factor)と呼ばれる精子形成領域が存在する.この領域の構造的特殊性を概観し,染色体内再組換えによる欠失機構を解説する.さらに,われわれが開発した日本人により適したY染色体微小欠失検出キットの開発のコンセプトとその使用法について概説する. Azoospermia is caused by abnormality in the various genes involved in spermatogenesis. However, the crucial genes for spermatogenesis have not yet been identified. Recently, targeted knockout and transgenic mice have been generated and considerable knowledge has been accumulated about their phenotype patterns, and a wide variety of genes are known to be associated with sperm formation. First, we review the meiotic and post-meiotic phases and genes expressed during spermatogenesis. Many genes corresponding to various clinical features of azoospermia exist, and we also review azoospermia related genes from clinical aspects. The AZF (azoospermia factor) regions on the Y chromosome long arm are thought to show a major correlation with spermatogenesis. From the genomic point of view, their deletion due to intrachromosomal recombimation is reviewed as a constitutional feature of the Y chromosome. Moreover, we explain how to use a detection kit for the Y chromosome micro deletion which we developed. 続きを見る
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論文

論文
高, 栄哲 ; Koh, Eitetsu
出版情報: 平成16(2004)年度 科学研究費補助金 基盤研究(C) 研究成果報告書 = 2004 Fiscal Year Final Research Report.  2003-2004  pp.4p.-,  2005-04. 
URL: http://hdl.handle.net/2297/00048935
概要: Y染色体のゲノム塩基配列が公開されると、相同性の高い反復配列とパリンドローム構造であることが明らかとなった。Y染色体上の大きなパリンドローム構造は8種あり、精子形成候補領域のひとつであるAZFcにはふたつのパリンドローム複合体が存在している 。我々は、パリンドローム単位で欠失が生じているという仮定のもとに、無・高度乏精子症患者ゲノムDNAを用いて、AZFcパリンドローム複合体の有無について検討した。まず、現在多用されている多コピー遺伝子や反復・繰返し配列の多いAZFc内部の欠失を確認することは従来のSTS-PCR法では理論的に不可能である。したがって、ゲノム塩基配列から、それぞれのプローブに対応するコピー数をreal-time quantitative PCR (RTQ PCR)によって確認し、特発性男性不妊症との関係について検討した。まず、定量化するために内部標準DNAを鋳型にして標準曲線を作成した。この鋳型は常染色体上の単一遺伝子であるRNAase P遺伝子を使用し、各サンプルのゲノムDNA量のばらつきを補正した。各患者サンプルの鋳型DNA量を10ngとし、RTQ-PCRを40サイクル行った。RTQ-PCRより算出できるコピー数比から、パリンドローム内部における微小欠失を検出することが可能となった。特に、パリンドローム部分欠失の検出は理論上無理とされていたが、ゲノムデータベースを基礎に男性不妊症でその部分欠失の存在を証明した。AZFc領域にあるP1+P2欠失のみならず、P1部分欠失の方法を確立した。今回使用したRTQ-PCR法において、従来のSTS-PCR法では欠失を認められない症例にも、AZFc領域内部に微小欠失が存在していることが明らかとなった。従来考えられていたよりも多くの特発性男性不妊症患者にAZFc領域の微小欠失が存在している可能性が考えられた。<br />Background :In 2003, the whole sequence of the Y chromosome has been determined following the completion of the human genome project. As a result, huge identical sequences have been found to be present massive Y-specific repeat called amplicons on the distal side of the euchromatic region of the long arm of the Y chromosome, and these amliconic regions have formed a massive palindrome complex. The palindromes in the AZFc region are consists of a complex of several small segments. These ampliconic sequences pairs are characterised by palindromes showing nearly more than 99.9% identy. Sequence tagged site (STS) markers are now available for detection of the position of palindromes. The AZFc region is comprised completely of amplicons and is particularly susceptible to deletion. The b2/b4 deletion is eliminated the entire AZFc region and cause the spermatogenetic failure. Some papers reported partial deletions within AZFc are present in some infertile male. Conventional PCR is not evaluat ed a copy number, but, real-time PCR assay that may be adaptable for estimating several identical sequence sites as copies numbers.The objective of the present study was to evaluate the multiple sites which is identical to sequences using the quantitative detection of copies by real-time fluorescence PCR as a new molecular diagnostic parameter in the genome DNA from patients presenting with non-obstructive azoospermia. Here we apply quantitative real-time PCR as an alternative approach to measure DNA copy number changes at each AZF region of the human Y chromosome.We selcted the probes such as sY 142 (G38345), sY 254 (G38349), sY 579 (G63909), sY 602 (G34986), sY 627 (G67175), sY 639 (G67162), sY 1054(G6716), sY 1125 (G67164), sY 1190 (G67165), sY 1192 (G67166), sY 1196 (G67167), sY 1197 (G67168), sY 1198 (G67169), sY 1201 (G67170), sY 1206 (G67171). We modified these primers size for real time PCR, so that the PCR products products is amplified approximately 100 base pairs and hybridization probes within each primer and dual-labelled flurogenic probes (hybridization probe) is designed between these selected modified primers. These amplicons and corresponding primers that showed a unique BLAST hit were selected and used in further experiments.The region of AZFc are present one or more identifical sequences per genome because of their palindromic structures. As more several retetive sequence exist, the possibility of partial deletion within AZFc is present.We here demonstrated the application of a new, flexible, fast, and precise real-time PCR based estimation the copy number of identical sequences in Y chromosome ampliconic region.<br />研究課題/領域番号:15591677, 研究期間(年度):2003-2004<br />出典:「特発性男性不妊症患者に対するY染色体パリンドローム複合体の分析と多型性検索」研究成果報告書 課題番号15591677(KAKEN:科学研究費助成事業データベース(国立情報学研究所))   本文データは著者版報告書より作成 続きを見る