Studies on the extracellular transporting mechanisms of sperm proteasomes and the functions of PA200

• Project/Area Number: 17K19520
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Biomedical structure and function and related fields
• Research Institution: Nagoya University
• Principal Investigator: Sawada Hitoshi 名古屋大学, 理学研究科, 教授 (60158946)
• Project Period (FY): 2017-06-30 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000); Fiscal Year 2018: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2017: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000)
• Keywords: プロテアソーム / 精子 / 受精 / PA200 / ユビキチン / 細胞外 / 酵素

Outline of Final Research Achievements

We previously reported that sperm proteasomes are involved in sperm penetration of the vitelline coat, probably as a lysin. Here, the localization of sperm proteasome in sperm membrane fraction was re-examined. Furthermore, roles in fertilization of proteasome activator PA200, which appears to be involved in sexual reproduction, was examined. The results showed that sperm proteasome subunits were detected by LC/MS in the immunoprecipitated sperm membrane fraction using anti-proteasome antibody. However, it was not successful to identify the proteasome-associating proteins. Then, targeting of ascidian PA200 genes was carried out by genome editing. However, this was not successful due to the induction of abnormal embryonic development. On the other hand, we noticed that sperm metalloproteases may be involved in ascidian fertilization based on the inhibitory effects on fertilization. This enzyme may also play a key role in fertilization together with the proteasome.

Academic Significance and Societal Importance of the Research Achievements

申請者は、受精実験が容易な脊索動物マボヤを用いて、その受精に精子ユビキチン-プロテアソーム系が関与すること、特に細胞外で卵膜ライシンとして機能することを初めて明らかにした。その後、哺乳類や鳥類や棘皮動物でも同様の現象が確認され、新口動物に共通するシステムであると考えられる。一般に、プロテアソームは細胞内のタンパク質分解で重要な役割を果たすが、細胞外での機能は不明な点が多い。しかし、近年、神経疾患等で細胞外プロテアソームが注目されつつある。本研究は、受精に関わる精子プロテアソームの細胞外での機能と輸送機構に焦点を当てた研究であり、不妊症診断治療薬の開発に繋がりうる基礎研究といえる。

Research Products

• [Journal Article] Three multi-allelic gene pairs are responsible for self-sterility in the ascidian Ciona intestinalis (2020)
  • Author(s): Sawada H, Yamamoto K, Yamaguchi A, Yamada L, Higuchi A, Nukaya H, Fukuoka M, Sakuma T, Yamamoto T, Sasakura Y, Shirae-Kurabayashi M
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 2514-2514
  • DOI: 10.1038/s41598-020-59147-4
  • Peer Reviewed / Open Access
• [Journal Article] Vitelline membrane proteins promote left-sided nodal expression after neurula rotation in the ascidian, Halocynthia roretzi. (2019)
  • Author(s): Tanaka, Y., Yamada, S., Connop, S. L., Hashii, N., Sawada, H., Shih, Y., and Nishida, H.
  • Journal Title: Dev. Biol. Volume: 449 Issue: 1 Pages: 52-61
  • DOI: 10.1016/j.ydbio.2019.01.016
  • Peer Reviewed
• [Journal Article] Peanut agglutinin specifically binds to a sperm region between the nucleus and mitochondria in tunicates and sea urchins. (2018)
  • Author(s): Nakazawa S, Shirae-Kurabayashi M, Sawada H.
  • Journal Title: Molecular Reproduction and Development Volume: 印刷中 Issue: 6 Pages: 464-477
  • DOI: 10.1002/mrd.22982
  • Peer Reviewed
• [Journal Article] Chitin-based barrier immunity and Its loss predated mucus-colonization by indigenous gut microbiota. (2018)
  • Author(s): Nakashima, K., Kimura, S., Ogawa, Y., Watanabe, S., Soma, S., Kaneko, T., Yamada, L., Sawada, H., Tung, C.-H., Lu, T-M., Yu, J-K., Briones, A. V., Kikuchi, S., and Satoh, N.
  • Journal Title: Nature Communications Volume: 9(1) Issue: 1 Pages: 3402-3402
  • DOI: 10.1038/s41467-018-05884-0
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants. (2018)
  • Author(s): Takahashi T., Mori, T., Ueda, K., Yamada, L., Nagahara, S., Higashiyama, T., Sawada, H., Igawa, T.
  • Journal Title: Development Volume: 145(23) Issue: 23
  • DOI: 10.1242/dev.170076
  • Peer Reviewed
• [Journal Article] Dual gene repertories for larval and adult shells reveal molecules for molluscan shell formation. (2018)
  • Author(s): Zhao, R., Takeuchi, T., Luo, Y-J., Ishikawa, A., Kobayashi, T., Koyanagi, R., Villar-Briones, A., Yamada, L., Sawada, H., Iwanaga, S., Nagai, K., Satoh, N., and Endo, K.
  • Journal Title: Molecular Biology and Evolution, Volume: 35(11) Pages: 2751-2761
  • DOI: 10.1093/molbev/msy172
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The role of metalloproteases in fertilisation in the ascidian Ciona robusta. (2018)
  • Author(s): Nakazawa, S., Shirae-Kurabayashi, M., and Sawada, H.
  • Journal Title: Scientific Reports, Volume: 9(1) Issue: 1 Pages: 1009-1009
  • DOI: 10.1038/s41598-018-37721-1
  • Peer Reviewed / Open Access
• [Journal Article] Ascidian sexual reproductive strategies: Mechanisms of sperm-egg interaction and self-sterility. (2018)
  • Author(s): Sawada H., Nakazawa, S., and Shirai-Kurabayashi, M.
  • Journal Title: Reproductive and Developmental Strategies: Diversity and Commonality in Animal (Springer Japan) Volume: 印刷中
  • Peer Reviewed
• [Journal Article] Haemorrhagic snake venom metalloproteases and human ADAMs cleave LRP5/6, which disrupts cell-cell adhesions in vitro and induces haemorrhage in vivo. (2017)
  • Author(s): Seo T, Sakon T, Nakazawa S, Nishioka A, Watanabe K, Matsumoto K, Akasaka M, Shioi N, Sawada H, Araki S
  • Journal Title: FEBS Journal Volume: 284 Issue: 11 Pages: 1657-1671
  • DOI: 10.1111/febs.14066
  • Peer Reviewed / Open Access
• [Journal Article] Spawning of the ascidian Halocynthia roretzi. (2017)
  • Author(s): Sawada, H., Shirae-Kurabayashi M., and Takaharu Numakunai
  • Journal Title: Molecular Reproduction and Development Volume: 84 Issue: 2 Pages: 83-83
  • DOI: 10.1002/mrd.22776
  • Peer Reviewed / Open Access
• [Journal Article] Isolation of GFP-tagged plasma membrane protein from Arabidopsis egg cells (2017)
  • Author(s): Igawa, T., Yamada, L., Sawada, H., Mori, T.
  • Journal Title: Plant Biotechnology Volume: 34 Issue: 2 Pages: 119-123
  • DOI: 10.5511/plantbiotechnology.17.0522a
  • NAID: 130006789291
  • ISSN: 1342-4580, 1347-6114
  • Peer Reviewed / Open Access
• [Presentation] Ascidian gamete self/nonself-recognition proteins (s-Themis and v-Themis) during fertilization of a hermaphroditic marine chordate Ciona intestinalis (2019)
  • Author(s): Hitoshi Sawada
  • Organizer: Gordon Research Conferences "Fertilization & Activation of Development"
  • Int'l Joint Research
• [Presentation] An Immunological Study of the Shell Matrix Protein ICP-1 in Brachiopods (2019)
  • Author(s): Yukinobu Isowa, Keiji Kito, Hitoshi Sawada and Kazuyoshi Endo
  • Organizer: 15th International Symposium on Biomineralization
  • Int'l Joint Research
• [Presentation] 雌雄同体のホヤが自家受精しないしくみと精子の卵膜通過について (2019)
  • Author(s): 澤田 均
  • Organizer: 第89回日本動物学会
  • Invited
• [Presentation] ホヤ精子アスタシン様金属プロテアーゼの性質と受精における役割 (2019)
  • Author(s): 服部 郁也， 橋下 志津弥，磯和 幸延， 白江-倉林 麻貴， 荒木 聡彦，澤田 均
  • Organizer: 第89回日本動物学会
• [Presentation] カタユウレイボヤの自家不和合性に関わるs/v-Themis-B2アレルのcDNA cloningと発現解析 (2019)
  • Author(s): 糠谷 晴彦， 白江-倉林 麻貴，澤田 均
  • Organizer: 第89回日本動物学会
• [Presentation] カタユウレイボヤの自家不和合性は低Ca2+海水により解除される (2019)
  • Author(s): 橋本 志津弥，澤田 均
  • Organizer: 第89回日本動物学会
• [Presentation] ホヤの受精機構：自家不和合性とライシンについて (2018)
  • Author(s): 澤田 均
  • Organizer: 日本動物学会
  • Invited
• [Presentation] 昆虫細胞を用いたカタユウレイボヤs-Themis, v-Themisタンパク質の相互作用解析 (2018)
  • Author(s): 成山 奏子、白江-倉林 麻貴、澤田 均
  • Organizer: 日本動物学会
• [Presentation] 昆虫細胞を用いたカタユウレイボヤs-Themis、v-Themisタンパク質の 相互作用についての研究 (2018)
  • Author(s): 成山 奏子 、西尾 俊亮 、白江-倉林 麻貴、松田 幹 、澤田 均
  • Organizer: 日本動物学会中部支部大会
• [Presentation] ADAM-LRP信号伝達経路に関わる新たな因子 (2018)
  • Author(s): 酒井 健有、澤田 均、荒木 聡彦
  • Organizer: 日本動物学会中部支部大会
• [Presentation] A method for isolation of specific Arabidopsis egg cell membrane protein without gamete isolation procedure. (2017)
  • Author(s): Tomoko Igawa, Lixy Yamada, Hitoshi Sawada, Toshiyuki Mori
  • Organizer: Cold Spring Harbor Asia Conference on Plant Cell & Development Biology
  • Int'l Joint Research
• [Presentation] Study on molecular players regulating male and female gamete interaction during double fertilization. (2017)
  • Author(s): Taro Takahashi, Toshiyuki Mori, Lixy Yamada, Hitoshi Sawada, Kenji Ueda, Shiori Nagahara, Tetsuya Higashiyama, Tomoko Igawa
  • Organizer: 第59回日本植物学会
• [Presentation] TALENを用いたカタユウレイボヤにおける精子タンパク質Urabinの機能解析 (2017)
  • Author(s): 成山奏子、白江-倉林麻貴、中澤志織、澤田均
  • Organizer: 第88回日本動物学会
• [Presentation] Roles of ascidian sperm metalloproteases in fertilization of a marine invertebrate Ciona robusta (2017)
  • Author(s): Shiori Nakazawa, Maki Shirae-Kurabayashi, Hitoshi Sawada
  • Organizer: Gordon Research Conference: Fertilization & Activation of Development
  • Int'l Joint Research
• [Presentation] cDNA cloning and expression of two ubiquitin-activating enzymes UBA1 and UBA6 in gametes of the ascidian Halocynthia rotezi (2017)
  • Author(s): Sawada, H., Inoue, S., Otsuka, K., Shirae-Kurabayashi
  • Organizer: Gordon Research Conference: Fertilization & Activation of Development
  • Int'l Joint Research
• [Presentation] Vascular endothelial cell-cell adhesion disruption and hemorrhage are induced by truncated LRP5/6 which is processed by hemorrhagic snake venom metalloproteases (2017)
  • Author(s): Seo, T., Kakon, T., Nakazawa, S., Nishioka, A., Watanabe, K., Matsumoto, K., Akasaka, M., Shioi, N., Sawada, H., and Araki, S.
  • Organizer: Gordon Research Conferences
  • Int'l Joint Research
• [Book] Reproductive in Aquatic Animals: From Basic Biology to Aquaculture Technology (M. Yoshida, and J. F. Asturiano eds.) (執筆担当Chapter 9; Self- and nonself-recognition of gametes in ascidians. pp.179-192) (2020)
  • Author(s): Sawada,H., Shirae-Kurabayashi, M. ほか
  • Total Pages: 379
  • Publisher: Springer
  • ISBN: 9789811522901
• [Book] Japanese Marine Life: A Practical Training Guide in Marine Biology (K. Inaba and J. Hall-Spencer, eds.) (執筆担当Gametes and fertilization, pp.93-107)) (2020)
  • Author(s): Inaba, k., Sawada, H., Yoshida, M., Shiba, K., Shirae-Kurabayashi, M. ほか
  • Total Pages: 367
  • Publisher: Springer
  • ISBN: 9789811513251
• [Book] Reproductive Biology and Aquaculture Technology in Aquatic Animals (Eds. Yoshida, M., Asturiano J. F.) (Chapter 9; Self- and nonself-recognition of gametes in ascidians.) (2019)
  • Author(s): Sawada, H. and Shirae-Kurabayashi, M.
  • Publisher: Springer
• [Book] Reproductive and Developmental Strategies: The Continuity of Life (Y. Iwao, S. Kobayashi eds.) (Chapter 23 Ascidian sexual reproductive strategies: Mechanisms of sperm-egg interaction and self-sterility.) (2018)
  • Author(s): Sawada H., Nakazawa, S., and Shirai-Kurabayashi, M ほか72人
  • Total Pages: 789
  • Publisher: Springer
• [Book] 動物学の百科事典（日本動物学会編） (2018)
  • Author(s): 澤田均 ほか308人
  • Total Pages: 800
  • Publisher: 丸善
  • ISBN: 9784621303092
• [Book] 動物の百科辞典（「受精」の章の執筆担当） (2018)
  • Author(s): 澤田均
  • Publisher: 丸善