Analysis of mammalian germ cell-specific GPI-anchored proteins using genetically modified animals

• Project/Area Number: 15H05573
• Research Category: Grant-in-Aid for Young Scientists (A)
• Allocation Type: Single-year Grants
• Research Field: Laboratory animal science
• Research Institution: Osaka University
• Principal Investigator: 藤原 祥高 大阪大学, 微生物病研究所, 招へい准教授 (70578848)
• Project Period (FY): 2015-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥23,400,000 (Direct Cost: ¥18,000,000、Indirect Cost: ¥5,400,000); Fiscal Year 2018: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2017: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2016: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2015: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000)
• Keywords: 哺乳類生殖細胞 / GPIアンカータンパク質 / 遺伝子組換えマウス / CRISPR/Cas9 / CRISPR/cas9 / ゲノム編集 / 点変異 / 精子受精能 / CRISPR/Cas9システム / ES細胞 / 生殖細胞 / 受精 / CRISPR/Casシステム

Outline of Annual Research Achievements

計画最終の今年度は、本課題の主プロジェクトの精子GPIアンカータンパク質LYPD4の機能を解析し、共通の表現型を持つ遺伝子（タンパク質）との関連を調べ、CRISPR/Cas9システムを用いたノックイン（KI）マウスを作製し検討を行った。その結果、前年度に論文発表（JCS. 2018）した精子膜タンパク質CMTM2A/BのKOマウスを用いてLYPD4との関連を調べたところ、LYPD4の発現に特に変化が見られなかったことから、LYPD4が既知の必須因子群とは別経路を介して精子受精能を制御している可能性を示唆する結果が得られた。また、これまで最も重要視されてきた精子膜タンパク質ADAM3（Exp Anim. 2018）との関連についても解析を行ったが、こちらもLypd4-KOマウスにおけるADAM3発現、及びAdam3-KOマウスにおけるLYPD4発現にも大きな影響が見られなかった。以上のことから、LYPD4が未知の経路を介して精子受精能を制御する新規タンパク質であることが明らかになった。これらの分子メカニズムを解明するために、LYPD4タンパク質のN末端側にFLAGタグを挿入したKIマウスの作製を試みた。その結果、作製には成功したが、残念ながらFLAG抗体を用いたWB及びIP-WB解析では目的タンパク質を検出することができなかった。現在、これまでの解析結果をまとめてLYPD4に関する論文として鋭意作成しており、準備ができ次第投稿する予定である。

Research Progress Status

平成30年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

平成30年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] Baylor College of Medicine(米国)
• [Int'l Joint Research] Baylor College of Medicine(アメリカ)
• [Int'l Joint Research] Baylor College of Medicine(アメリカ)
• [Journal Article] Seminal vesicle secretory protein 7, PATE4, is not required for sperm function but for copulatory plug formation to ensure fecundity† (2019)
  • Author(s): Noda Taichi、Fujihara Yoshitaka、Matsumura Takafumi、Oura Seiya、Kobayashi Sumire、Ikawa Masahito
  • Journal Title: Biology of reproduction Volume: 100 Pages: 1035-1045
  • DOI: 10.1093/biolre/ioy247
  • Peer Reviewed / Open Access
• [Journal Article] Co-expression of sperm membrane proteins CMTM2A and CMTM2B is essential for ADAM3 localization and male fertility in mice. (2018)
  • Author(s): Fujihara Y, Oji A, Kojima-Kita K, Larasati T, Ikawa M.
  • Journal Title: Journal of Cell Science Volume: 131 Pages: 221481-221481
  • DOI: 10.1242/jcs.221481
  • Peer Reviewed / Open Access
• [Journal Article] MAFB is dispensable for the fetal testis morphogenesis and the maintenance of spermatogenesis in adult mice. (2018)
  • Author(s): S. Sakamoto, D. Thumkeo, H. Ohta, Z. Zhang, S.R. Huang, P. Kanchenawong, T. Fuu, S. Watanabe, K. Shimada, Y. Fujihara, S. Yoshida, M. Ikawa, N. Watanabe, M. Saitou and S. Narumiya
  • Journal Title: PLoS Biol. Volume: 16
  • DOI: 10.1371/journal.pbio.2004874
  • NAID: 120006410921
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] MARCKSL1 Regulates Spine Formation in the Amygdala and Controls the Hypothalamic-Pituitary-Adrenal Axis and Anxiety-Like Behaviors. (2018)
  • Author(s): Tanaka T, Shimizu S, Ueno M, Fujihara Y, Ikawa M, Miyata S.
  • Journal Title: EBioMedicine Volume: 30 Pages: 62-73
  • DOI: 10.1016/j.ebiom.2018.03.018
  • Peer Reviewed / Open Access
• [Journal Article] Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn's Disease. (2018)
  • Author(s): Kurahara Lin Hai、Hiraishi Keizo、Hu Yaopeng、Koga Kaori、Onitsuka Miki、Doi Mayumi、Aoyagi Kunihiko、Takedatsu Hidetoshi、Kojima Daibo、Fujihara Yoshitaka、Jian Yuwen、Inoue Ryuji
  • Journal Title: Cellular and molecular gastroenterology and hepatology Volume: 5 Pages: 299-318
  • DOI: 10.1016/j.jcmgh.2017.12.005
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Factors controlling sperm migration through the oviduct revealed by gene-modified mouse models (2018)
  • Author(s): fujihara Y, Miyata H, Ikawa M.
  • Journal Title: Exp. Anim. Volume: - Issue: 2 Pages: 91-104
  • DOI: 10.1538/expanim.17-0153
  • NAID: 130006733631
  • Peer Reviewed / Open Access
• [Journal Article] Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a. (2018)
  • Author(s): Higa M, Oka M, Fujihara Y, Masuda K, Yoneda Y, Kishimoto T.
  • Journal Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 6:115(6)
  • DOI: 10.1073/pnas.1719921115
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Human globozoospermia-rclatcd gcne spata16 ls required for sperm formation revcaled by CRISPR/Cas9-mediatcd mouse modcls. (2017)
  • Author(s): Fujihara Y, Oji A, Larasati T, Kojima-Kita K, Ikawa M.
  • Journal Title: International Journal of Molecular Sciences Volume: 18 Pages: 2208-2208
  • DOI: 10.3390/ijms18102208
  • Peer Reviewed / Open Access
• [Journal Article] JunB is essential for IL-23-dependent pathogenicity of Th17 cells (2017)
  • Author(s): Hasan Z, Koizumi SI, Sasaki D, Yamada H, Arakaki N, Fujihara Y, Okitsu S, Shirahata H, Ishikawa H.
  • Journal Title: Nature Communications Volume: 8 Pages: 15628-15628
  • DOI: 10.1038/ncomms15628
  • Peer Reviewed / Open Access
• [Journal Article] A chromosome 16p13.11 microduplication causes hyperactivity through dysregulation of miR-484/protocadherin-19 signaling (2017)
  • Author(s): Fujitani, M., Zhang, S., Fujiki, R., Fujihara, Y. and Yamashita, T.
  • Journal Title: Molecular Psychiatry Volume: 22 Pages: 364-374
  • DOI: 10.1038/mp.2016.106
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A GPI processing phospholipase A2, PGAP6, modulates Nodal signaling in embryos by shedding CRIPTO. (2016)
  • Author(s): Lee GH, Fujita M, Takaoka K, Murakami Y, Fujihara Y, Kanzawa N, Murakami KI, Kajikawa E, Takada Y, Saito K, Ikawa M, Hamada H, Maeda Y, Kinoshita T.
  • Journal Title: The Journal of Cell Biology Volume: 215 Pages: 705-718
  • Peer Reviewed
• [Journal Article] CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice (2016)
  • Author(s): Oji A, Noda T, Fujihara Y, Miyata H, Kim YJ, Muto M, Nozawa K, Matsumura T, Isotani A, Ikawa M.
  • Journal Title: Scientific Reports Volume: 6 Pages: 31666-31666
  • DOI: 10.1038/srep31666
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Live imaging of X chromosome reactivation dynamics in early mouse development can discriminate naive from primed pluripotent stem cells (2016)
  • Author(s): Kobayashi S, Hosoi Y, Shiura H, Yamagata K, Takahashi S, Fujihara Y, Kohda T, Okabe M, Ishino F.
  • Journal Title: DEVELOPMENT Volume: 143 Pages: 2958-2964
  • DOI: 10.1242/dev.136739
  • Peer Reviewed / Open Access
• [Journal Article] Structural and functional insights into IZUMO1 recognition by JUNO in mammalian fertilization. (2016)
  • Author(s): Kato K, Satouh Y, Nishimasu H, Kurabayashi A, Morita J, Fujihara Y, Oji A, Ishitani R, Ikawa M, Nureki O.
  • Journal Title: Nat. Commun. Volume: 7 Pages: 12198-12198
  • DOI: 10.1038/ncomms12198
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice. (2016)
  • Author(s): Miyata H, Castaneda JM, Fujihara Y, Yu Z, Archambeault DR, Isotani A, Kiyozumi D, Kriseman ML, Mashiko D, Matsumura T, Matzuk RM, Mori M, Noda T, Oji A, Okabe M, Prunskaite-Hyyrylainen R, Ramirez-Solis R, Satouh Y, Zhang Q, Ikawa M, Matzuk MM.
  • Journal Title: Proc Natl Acad Sci USA. Volume: 113 Issue: 28 Pages: 7704-10
  • DOI: 10.1073/pnas.1608458113
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] The Mg2+ transporter CNNM4 regulates sperm Ca2+ homeostasis and it is essential for reproduction. (2016)
  • Author(s): Yamazaki D, Miyata H, Funato Y, Fujihara Y, Ikawa M, Miki H.
  • Journal Title: J Cell Sci. Volume: 未定 Issue: 9 Pages: 19401949-19401949
  • DOI: 10.1242/jcs.182220
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] GPI-AP release in cellular, developmental, and reproductive biology. (2016)
  • Author(s): Fujihara Y, Ikawa M.
  • Journal Title: J Lipid Res. Volume: 57 Issue: 4 Pages: 538-45
  • DOI: 10.1194/jlr.r063032
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 生殖不全の表現型解析 (2016)
  • Author(s): 藤原祥高，佐藤裕公，伊川正人
  • Journal Title: 実験医学別冊マウス表現型解析スタンダード Volume: 1 Pages: 216-227
• [Journal Article] Lentiviral Vector-Mediated Complementation Restored Fetal Viability but Not Placental Hyperplasia in Plac1-Deficient Mice (2016)
  • Author(s): Muto M, Fujihara Y, Tobita T, Kiyozumi D, Ikawa M.
  • Journal Title: Biology of Reproduction Volume: 94(1) Pages: 6-6
  • DOI: 10.1095/biolreprod.115.133454
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Sperm calcineurin inhibition prevents mouse fertility with implications for male contraceptive. (2015)
  • Author(s): Haruhiko Miyata, Yuhkoh Satouh, Daisuke Mashiko, Masanaga Muto, Kaori Nozawa, Kogiku Shiba, Yoshitaka Fujihara, Ayako Isotani, Kazuo Inaba, Masahito Ikawa
  • Journal Title: Science. Volume: 350 Pages: 442-445
  • DOI: 10.1126/science.aad0836
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Calreticulin is required for development of the cumulus oocyte complex and female fertility (2015)
  • Author(s): Tokuhiro K, Satouh Y, Nozawa K, Isotani A, Fujihara Y, Hirashima Y, Matsumura H, Takumi K, Miyano T, Okabe M, Benham AM, Ikawa M.
  • Journal Title: Sci Rep Volume: 5 Pages: 14254-14254
  • DOI: 10.1038/srep14254
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes (2015)
  • Author(s): Ono R., Ishii M., Fujihara Y., Kitazawa M., Usami T., Kaneko-Ishino T., Kanno J., Ikawa M., Ishino F.
  • Journal Title: Sci Rep Volume: 5 Pages: 12281-12281
  • DOI: 10.1038/srep12281
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] 第II部第3章 マウスでのゲノム編集 (2015)
  • Author(s): 藤原祥高、伊川正人
  • Journal Title: ゲノム編集成功の秘訣Q＆A Volume: 1 Pages: 106-123
• [Presentation] ゲノム編集動物作製法の開発と雄性不妊モデルマウスの機能解析 (2019)
  • Author(s): 藤原 祥高
  • Organizer: 第19回日本生殖工学会学術講演会
  • Invited
• [Presentation] Human globozoospermia-related gene Spata16 is required for sperm formation revealed by CRISPR/Cas9-mediated mouse models (2019)
  • Author(s): Yoshitaka Fujihara, Asami Oji, Tamara Larasati, Kanako Kojima-Kita, and Masahito Ikawa
  • Organizer: 66th Annual Meeting of the Society for Reproductive Investigation
  • Int'l Joint Research
• [Presentation] 遺伝子改変マウスを用いた精巣特異的Cmtm遺伝子群の機能解析 (2018)
  • Author(s): 藤原 祥高、大字 亜沙美、北 加奈子、Tamara Larasati、伊川 正人
  • Organizer: 第65回日本実験動物学会総会
• [Presentation] Human globozoospermia-related gene Spata16 is required for sperm formation revealed by CRISPR/Cas9-mediated mouse models (2018)
  • Author(s): Yoshitaka Fujihara, Asami Oji, Tamara Larasati, Kanako Kojima-Kita, and Masahito Ikawa
  • Organizer: 51st Annual Meeting of the Society for the Study of Reproduction
  • Int'l Joint Research
• [Presentation] Generation of point mutant mouse model responsible for human globozoospermia by CRISPR/Cas9 system (2017)
  • Author(s): Yoshitaka Fujihara, Asami Oji, Kanako Kita, and Masahito Ikawa
  • Organizer: 第64回日本実験動物学会総会
• [Presentation] 遺伝子改変技術を用いた雄性不妊モデルマウスの開発と機能解析 (2017)
  • Author(s): 藤原祥高
  • Organizer: 第64回日本実験動物学会総会
  • Invited
• [Presentation] 遺伝子改変マウスを用いた雄性生殖細胞特異的な膜タンパク質の機能解析 (2017)
  • Author(s): 藤原祥高
  • Organizer: 奈良先端科学技術大学院大学バイオサイエンス研究科セミナー
  • Invited
• [Presentation] Human globozoospermia-related gene Spata16 is essential for spermiogenesis in mice (2017)
  • Author(s): Yoshitaka Fujihara, Asami Oji, Tamara Larasati, Kanako Kojima-Kita and Masahito Ikawa
  • Organizer: Fourth World Congress of Reproductive Biology
  • Int'l Joint Research
• [Presentation] 遺伝子改変技術を用いた雄性不妊モデルマウスの開発と機能解析 (2017)
  • Author(s): 藤原祥高
  • Organizer: 第14回熊本大学生命資源研究・支援センターシンポジウム
  • Invited
• [Presentation] Human globozoospermia-related gene Spata16 is essential for spermiogenesis in mice (2017)
  • Author(s): Yoshitaka Fujihara, Asami Oji, Tamara Larasati, Kanako Kojima-Kita and Masahito Ikawa
  • Organizer: 2017年度生命科学系学会合同年次大会（第40回日本分子生物学会年会）
• [Presentation] GPI-anchored protein complex, TEX101 and LY6K, is required for sperm fertilizing ability in mice (2016)
  • Author(s): Yoshitaka Fujihara, Masaru Okabe, Masahito Ikawa
  • Organizer: 国際シンポジウム "生殖細胞のエピゲノムダイナミクスとその制御"
  • Place of Presentation: 京都大学百周年時計台記念館
  • Year and Date: 2016-02-17
  • Int'l Joint Research
• [Presentation] 雄性生殖細胞特異的 GPI アンカータンパク質の機能解析と新規遺伝子改変マウス作製法の開発 (2015)
  • Author(s): 藤原祥高
  • Organizer: 神戸大学重点研究チーム「資源動物のシグナル伝達制御に関する研究」 ワークショップ 「生殖細胞研究の最先端技術」
  • Place of Presentation: 神戸大学農学研究科
  • Year and Date: 2015-09-11
  • Invited
• [Presentation] GPI-anchored protein complex, LY6K/TEX101, is required for sperm fertilizing ability in mice (2015)
  • Author(s): Yoshitaka Fujihara, Masaru Okabe, Masahito Ikawa
  • Organizer: 第10回研究所ネットワーク国際シンポジウム
  • Place of Presentation: 北海道大学遺伝子病制御研究所
  • Year and Date: 2015-07-23
  • Int'l Joint Research
• [Presentation] 雄性生殖細胞特異的な発現を示すGPIアンカータンパク質複合体LY6K/TEX101の精子受精能における役割 (2015)
  • Author(s): 藤原祥高、岡部勝、伊川正人
  • Organizer: 第62回日本実験動物学会総会
  • Place of Presentation: 京都テルサ
  • Year and Date: 2015-05-29
• [Remarks] researchmap
  • URL: https://researchmap.jp/Yoshitaka_Fujihara/
• [Remarks] ORCID
  • URL: https://orcid.org/0000-0001-8332-3507
• [Remarks] 大阪大学 研究者総覧
  • URL: http://www.dma.jim.osaka-u.ac.jp/view?l=ja&u=6639
• [Remarks] 大阪大学微生物病研究所 遺伝子機能解析分野 メンバー紹介ページ
  • URL: http://www.egr.biken.osaka-u.ac.jp/information/yoshitaka_fujihara.html