Structure and function of spermatocyte nucleolus in the meiotic prophase; electron microscopic and molecular anatomical analysis

• Project/Area Number: 20K07246
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 48010:Anatomy-related
• Research Institution: Nagasaki University
• Principal Investigator: Koji Takehiko 長崎大学, 医歯薬学総合研究科(医学系), 客員研究員 (30170179)
• Co-Investigator(Kenkyū-buntansha): 柴田 恭明 長崎大学, 医歯薬学総合研究科(医学系), 准教授 (80253673) ; 末松 貴史 長崎大学, 医歯薬学総合研究科(医学系), 技術職員 (70264249)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2022: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2021: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2020: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: マウス / 精巣 / DNAメチル化 / 5-aza-2’-deoxycitidine / 核小体 / 精祖細胞 / 精母細胞 / セルトリ細胞 / nucleolin / 精原細胞 / rRNA / In situ PCR / 5-azadC / pre-rRNA及びrRNAs / 電顕ISH / ホスホグリセリン酸キナーゼ遺伝子局在 / 構造と機能 / エピゲノム / アポトーシス

Outline of Research at the Start

哺乳類精子形成過程減数分裂前期に残存する核小体が染色体配置に重要な役割を果たす可能性がある。本研究ではマウス精巣に於いて、減数分裂前期でのrDNA位置の同定やrRNAの産生・分布及びリボソームサブユニット形成動態を電顕in situ hybridization (ISH)法と超解像顕微鏡を駆使して解明すると共に、減数分裂時にX染色体のPgk-1から17番染色体のPgk-2に発現が切り替わるホスホグリセリン酸キナーゼ遺伝子局在と発現状態を解析し、ヘテロクロマチン化に関し核小体との関係を検討する。また、DNAのメチル化及びヒストン修飾等の核小体機能と染色体の核内配置調節への関与を明らかにする。

Outline of Final Research Achievements

The nucleoli of spermatocytes are very unique and partially maintained during prophase of meiosis, while that of round spermatid is conspicuous but without rRNA localization. For better understanding of structure and function of the nucleoli, we conducted molecular histochemical analyses of the expression of rRNA in normal and DNA methylation inhibitor, 5-aza-2’-deoxycytidine (5-azadC) treated adult mouse testes. As a result, the transcription activity of rRNA in the nucleoli of round spermatids was suppressed because of high level of methylation of rDNA, in consistent with no nucleolin spots. The decrease in the level of DNA methylation by 5-azadC increased the number of nucleoli as well as nucleolin spots in spermatocytes. These results indicate that the states of spermatogenic cell nucleoli are regulated epigenetically by DNA methylation level of rDNA, which is progressively increased during mouse spermatogenesis.

Academic Significance and Societal Importance of the Research Achievements

核小体は核膜周囲と連なるヘテロクロマチンに囲まれ、クロマチン凝集の起点として、また染色体領域を決める核内極性の基軸として働く可能性が指摘されているが、その形成過程の詳細は不明である。本研究では精子形成過程における核小体の不活化がDNAメチル化によって誘導されることを明らかにした。人為的rRNA 転写制御は、核小体形成過程の解明や、がん悪性度の指標となる核小体数の増加機序の解明に寄与すると期待される。

Research Products

• [Journal Article] Transcriptome proiling of anhidrotic eccrine sweat glands reveals that Olfactory receptors on eccrine sweat glands regulate perspiration in a ligand-dependent manner, (2023)
  • Author(s): Murayama N, Miyaki T, Okuzaki D, Shibata Y, Koji T, Inoue A, Aoki J, Hayashi H, Tanaka Y, Murota H
  • Journal Title: JID Innov Volume: - Issue: 4 Pages: 100196-100196
  • DOI: 10.1016/j.xjidi.2023.100196
  • Peer Reviewed / Open Access
• [Journal Article] The transfer of the Hepatocyte Growth Factor gene by macrophages ameliorates the progression of peritoneal fibrosis in mice. (2023)
  • Author(s): Obata Y, Abe K, Miyazaki M, Koji T, Tabata Y, Nishino T
  • Journal Title: Int J Mol Sci Volume: 24 Issue: 8 Pages: 6951-6951
  • DOI: 10.3390/ijms24086951
  • Peer Reviewed / Open Access
• [Journal Article] An Advanced Detection System for <i>In Situ</i> Hybridization Using a Fluorescence Resonance Energy Transfer-based Molecular Beacon Probe (2022)
  • Author(s): Narantsog Choijookhuu, Yasuaki Shibata, Takumi Ishizuka, Yan Xu, Takehiko Koji, Yoshitaka Hishikawa
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 55 Issue: 5 Pages: 119-128
  • DOI: 10.1267/ahc.22-00075
  • ISSN: 0044-5991, 1347-5800
  • Year and Date: 2022-10-28
  • Peer Reviewed / Open Access
• [Journal Article] Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD. (2022)
  • Author(s): Hattori N, Nakagawa T, Yoneda M, Hayashida H, Nakagawa K, Yamamoto K, Myo Win Htun, Shibata Y, Koji T, Ito T
  • Journal Title: J Biochem Volume: 172 Issue: 6 Pages: 365-376
  • DOI: 10.1093/jb/mvac077
  • Peer Reviewed / Open Access
• [Journal Article] Hydroxychloroquine suppresses anti-GBM nephritis via inhibition of JNK/p38 MAPK signaling (2022)
  • Author(s): Torigoe Miki、Obata Yoko、Inoue Hiro、Torigoe Kenta、Kinoshita Akira、Koji Takehiko、Mukae Hiroshi、Nishino Tomoya
  • Journal Title: Clinical and Experimental Nephrology Volume: 27 Issue: 2 Pages: 110-121
  • DOI: 10.1007/s10157-022-02285-y
  • Peer Reviewed / Open Access
• [Journal Article] Mitochonic acid-5 ameliorates chlorhexidine gluconate-induced peritoneal fibrosis in mice. (2022)
  • Author(s): Inoue H, Torigoe K, Torigoe M, Muta K, Obata Y, Suzuki T, Suzuki C, Abe T, Koji T, Mukae H, Nishino T
  • Journal Title: Med Mol Morphol Volume: 55 Issue: 1 Pages: 27-40
  • DOI: 10.1007/s00795-021-00305-6
  • Peer Reviewed / Open Access
• [Journal Article] In focus in HCB: new histochemical insights into mammalian gametogenesis. (2022)
  • Author(s): Hishikawa Y, Takizawa T, Koji T
  • Journal Title: Histochem Cell Biol Volume: 157 Issue: 3 Pages: 269-271
  • DOI: 10.1007/s00418-022-02083-3
  • Peer Reviewed / Open Access
• [Journal Article] Pathological differences in the bone healing processes between tooth extraction socket and femoral fracture (2022)
  • Author(s): Ito Shinichirou、Kasahara Norio、Kitamura Kei、Matsunaga Satoru、Mizoguchi Toshihide、Htun Myo Win、Shibata Yasuaki、Abe Shinichi、Takano Masayuki、Yamaguchi Akira
  • Journal Title: Bone Reports Volume: 16 Pages: 101522-101522
  • DOI: 10.1016/j.bonr.2022.101522
  • Peer Reviewed / Open Access
• [Journal Article] Nuclear Expression of Pygo2 Correlates with Poorly Differentiated State Involving c-Myc, PCNA and Bcl9 in Myanmar Hepatocellular Carcinoma (2021)
  • Author(s): Myo Win Htun, Shibata Y, Kyaw Soe, Koji T
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 54 Issue: 6 Pages: 195-206
  • DOI: 10.1267/ahc.21-00090
  • NAID: 130008132654
  • ISSN: 0044-5991, 1347-5800
  • Year and Date: 2021-12-24
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] An inhibitor of Kr&uuml;ppel-like factor 5 suppresses peritoneal fibrosis in mice. (2021)
  • Author(s): Muta K, Nakazawa Y, Obata Y, Inoue H, Torigoe K, Nakazawa M, Abe K, Furusu A, Miyazaki M, Yamamoto K, Koji T, Nishino T
  • Journal Title: Perit Dial Int Volume: - Issue: 4 Pages: 394-403
  • DOI: 10.1177/0896860820981322
  • Peer Reviewed / Open Access
• [Journal Article] Vitamin K-dependent γ-glutamyl carboxylase in Sertoli cells is essential for male fertility in mice. (2021)
  • Author(s): Sachiko Shiba, Kazuhiro Ikeda, Kuniko Horie-Inoue, Kotaro Azuma, Tomoka Hasegawa, Norio Amizuka, Tomoaki Tanaka, Toshihiko Takeiwa, Yasuaki Shibata, Takehiko Koji, Satoshi Inoue
  • Journal Title: Mol Cell Biol. Volume: 41(4) Issue: 4 Pages: 404-20
  • DOI: 10.1128/mcb.00404-20
  • Peer Reviewed / Open Access
• [Journal Article] Global changes in epigenomes during mouse spermatogenesis: possible relation to germ cell apoptosis. (2020)
  • Author(s): Koji T, Shibata Y
  • Journal Title: Histochem Cell Biol Volume: 154 Issue: 2 Pages: 123-134
  • DOI: 10.1007/s00418-020-01900-x
  • Peer Reviewed / Open Access
• [Journal Article] Hexapeptide derived from prothymosin alpha attenuates cisplatin-induced acute kidney injury. (2020)
  • Author(s): Torigoe K, Obata Y, Torigoe M, Oka S, Yamamoto K, Koji T, Ueda H, Mukae H, Nishino T
  • Journal Title: Clin Exp Nephrol Volume: 24 Issue: 5 Pages: 411-419
  • DOI: 10.1007/s10157-019-01843-1
  • Peer Reviewed / Open Access
• [Presentation] Myanmar人肝細胞がんに於けるWnt/β-catenin補助因子Pygo2とｃ－Mycの相関発現。 (2023)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第128回日本解剖学会総会・全国学術集会
• [Presentation] Differentiation-dependent changes in epigenetic factors in mouse spermatogenesis and their possible relations to germ cell apoptosis (HCB Lecture) . (2022)
  • Author(s): Koji T
  • Organizer: 16th International Congress on Histochemistry and Cytochemistry
  • Invited
• [Presentation] Myanmar人肝細胞がんに於けるPygo2 高発現とBcl9, c-Mye, PCNAの相関発現。 (2022)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第63回日本組織細胞化学会総会・学術集会
• [Presentation] 生命規範の新たな展望：エピゲノムによる分化制御と人為操作。 (2022)
  • Author(s): 小路武彦
  • Organizer: 第54回日本臨床分子形態学会総会・学術集会
  • Invited
• [Presentation] ミャンマー人肝細胞がんに於けるWnt/β-cateninシグナル補助因子と c-Mycの発現相関。 (2022)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第54回日本臨床分子形態学会総会・学術集会
• [Presentation] ミャンマー人肝細胞がん分化型低下に伴うPygo2及びc-Mycの相関的な発現上昇。 (2022)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第127回日本解剖学会総会・全国学術集会
• [Presentation] 蛍光共鳴エネルギー移動（FRET）を利用したin situ hybridizationの実際。 (2021)
  • Author(s): 菱川善隆、Choijookhuu N、石塚匠、柴田恭明、小路武彦
  • Organizer: 第62回日本組織細胞化学会総会・学術集会
• [Presentation] Nuclear expression of Pygo2 and Bcl9 complex correlates that of c-Myc and PCNA in Myanmar hepatocellular carcinoma. (2021)
  • Author(s): Myo Win Htun, Shibata Y, Koji T
  • Organizer: 第53回日本臨床分子形態学会総会・学術集会
• [Presentation] 顕微解析対象としての生命科学の根本規範：ゲノムからエピゲノムへ。 (2021)
  • Author(s): 小路武彦
  • Organizer: 日本顕微鏡学会第64回シンポジウム
  • Invited
• [Presentation] In situ PCRを用いたマウス雄性生殖細胞に於けるPgk-1、-2遺伝子のinter-chromosomal interaction解析。 (2021)
  • Author(s): 柴田恭明、小路武彦
  • Organizer: 第126回日本解剖学会総会・全国学術集会
• [Presentation] 超解像顕微鏡による細胞内タンパク質凝集体の形態解析。 (2020)
  • Author(s): 松本弦、柴田恭明、小路武彦
  • Organizer: 第62回日本顕微鏡学会九州支部集会・学術講演会
• [Presentation] Localization of c-Myc, CD163 and NF-κB positive macrophages in the iron overload rat liver after partial hepatectomy. (2020)
  • Author(s): Myo Win Htun, Shibata Y, Koji T
  • Organizer: 第62回日本顕微鏡学会九州支部集会・学術講演会
• [Presentation] 2つのBcl9抗体を用いたミャンマー国肝細胞がんの分別的免疫組織化学：若年性肝細胞がんにおける細胞内Bcl9発現の低下。 (2020)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第62回日本顕微鏡学会九州支部集会・学術講演会
• [Presentation] DES依存性マウス下垂体ゴナドトロフからPRL細胞への分化転換にはヒストンH3K9の脱アセチル化が関与する。 (2020)
  • Author(s): 柴田恭明、Myo Win Htun、小路武彦
  • Organizer: 第52回日本臨床分子形態学会総会・学術集会
• [Presentation] 分子組織細胞化学の開拓と展開。 (2020)
  • Author(s): 小路武彦
  • Organizer: 第61回日本組織細胞化学会総会・学術集会
  • Invited
• [Presentation] The two Bcl9 antibodies that recognize different epitopes immunohistochemically characterize juvenile development of HCC in Myanmar. (2020)
  • Author(s): Myo Win Htun, Myat Thu Soe, Shibata Y, Abe K, Koji T
  • Organizer: 第61回日本組織細胞化学会総会・学術集会
• [Presentation] Hydroxychloroquine prevents anti-GBM glomerulonephritis in rats. (2020)
  • Author(s): Torigoe M, Obata Y, Inoue H, Torigoe K, Kinoshita A, Koji T, Nishino T
  • Organizer: 57th ERA-EDTA Congress
  • Int'l Joint Research
• [Presentation] Mitochonic acid 5(MA-5) ameliorates Cg-induced peritoneal fibrosis in mice. (2020)
  • Author(s): Inoue H, Obata Y, Suzuki T, Torigoe M, Torigoe K, Suzuki C, Abe T, Koji T, Nishino T
  • Organizer: 57th ERA-EDTA Congress
  • Int'l Joint Research
• [Presentation] Mitochonic acid 5 alleviates chlorhexidine gluconate-induced peritoneal fibrosis in mice. (2020)
  • Author(s): Inoue H, Obata Y, Suzuki T, Torigoe M, Torigoe K, Muta K, Suzuki C, Abe T, Koji T, Nishino T
  • Organizer: ASN KIDNEY WEEK 2020
  • Int'l Joint Research
• [Presentation] ヒドロキシクロロキンは抗GBM抗体腎炎の進展を抑制する。 (2020)
  • Author(s): 鳥越未来、小畑陽子、井上大、鳥越健太、木下晃、小路武彦、西野友哉
  • Organizer: 第63回日本腎臓学会学術総会
• [Presentation] Mitochonic Acid 5(MA-5)はCG 誘発性マウス腹膜線維化を抑制する。 (2020)
  • Author(s): 井上大、小畑陽子、鈴木健弘、鳥越未来、鳥越健太、鈴木千登世、阿部高明、小路武彦、西野友哉
  • Organizer: 第63回日本腎臓学会学術総会
• [Book] 組織細胞化学 (2022) (2022)
  • Author(s): 菱川善隆、柴田恭明、小路武彦
  • Total Pages: 258
  • Publisher: 学祭企画
  • ISBN: 4906514979