Studies on functions of membrane steroid receptors and molecular mechanism of oocyte maturation and ovulation

• Project/Area Number: 16K07419
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Morphology/Structure
• Research Institution: Shizuoka University
• Principal Investigator: TOKUMOTO Toshinobu 静岡大学, 創造科学技術大学院, 教授 (30273163)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: ステロイド膜受容体 / 卵成熟 / 排卵 / 遺伝子発現 / 蛋白質 / ホルモン受容体

Outline of Final Research Achievements

We established quadruple mutant strain of membrane progestin receptors in Medaka fish. The mutant stain showed defect in their fecundity.
We selected 11 candidate genes for induction of ovulation in zebrafish. Then we established gene knock-out strains of these genes by CRISPR/Cas9 system. Among them, starmaker (stm) gene knock-out strain showed low fertilization late and abnormal embryos were developed. The results demonstrated that stm gene possesses the roles in fertilization in addition to formation of ear stones. Pax2a gene knock-out strain also showed low fertilization rate and abnormal development. Pax2a is known to have a important roles in early development but also have function during on the beginning of development.

Academic Significance and Societal Importance of the Research Achievements

ステロイド膜受容体（mPR）4重変異体メダカで見られた妊性の低下は初めての知見となる。ステロイド膜受容体を介したステロイドホルモンの作用が生理学的意味を持つことを示す実験的証拠と成り得る。今後、卵巣の細胞レベルでの観察などにより詳細を調べる。さらにゼブラフィッシュを用いたゲノム編集技術によるmPR遺伝子群のゲノム編集系統を樹立し、確証を得る。排卵誘導遺伝子候補については二つの遺伝子について受精前後の新たな機能を示すことができた。引き続き残りのゲノム編集系統の表現型解析を進め、排卵誘導の鍵遺伝子の解明を目指す。

Research Products

• [Int'l Joint Research] モンクット王工科大学ラートクラバン校(タイ)
• [Journal Article] Candidate gene identification of ovulation- inducing genes by RNA sequencing with an in vivo assay in zebrafish. (2018)
  • Author(s): W. Klangnurak, T. Fukuyo, MD. Rezanujjaman, M. Seki, S. Sugano, Y. Suzuki, T. Tokumoto
  • Journal Title: PLOS ONE Volume: 13 Issue: 5 Pages: 1-19
  • DOI: 10.1371/journal.pone.0196544
  • NAID: 120006603025
  • Peer Reviewed / Open Access
• [Journal Article] <b>Characterization of membrane progestin receptor α (mPRα) of the medaka and role in the induction of oocyte </b><b>maturation </b> (2017)
  • Author(s): Shimi Rani Roy, Jun Wang, Mikiko Nakashima, Toshinobu Tokumoto
  • Journal Title: Biomedical Research Volume: 38 Issue: 1 Pages: 79-87
  • DOI: 10.2220/biomedres.38.79
  • NAID: 130005242918
  • ISSN: 0388-6107, 1880-313X
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Fine selection of up-regulated genes during ovulation by in vivo induction of oocyte maturation and ovulation in zebrafish. (2017)
  • Author(s): Wanlada Klangnurak, Toshinobu Tokumoto
  • Journal Title: Zoological Letters Volume: 3
  • NAID: 120006603023
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Role of Starmarker (stm) Gene for Fertilization in Zebrafish (2019)
  • Author(s): Taketo Fukuyo, Wanlada Klangnurak, Md.Rezanujjaman, Toshinobu Tokumoto
  • Organizer: 2019 9th International Conference on Bioscience, Biochemistry and Bioinformatics (ICBBB2019) at Singapore
  • Int'l Joint Research
• [Presentation] Establishment of Gene Knock-out Strain of pax2a Selected as an Ovulation-inducing Gene by in vivo Assay by Genome Editing in Zebrafish (2019)
  • Author(s): Theeranukul Pachoensuk, Fukuyo Taketo, Klangnurak Wanlada, Toshinobu Tokumoto
  • Organizer: 2019 9th International Conference on Bioscience, Biochemistry and Bioinformatics (ICBBB2019) at Singapore
  • Int'l Joint Research
• [Presentation] IDENTIFICATION OF OVULATION-INDUCING GENES SELECTED BY IN VIVO ASSAY BY GENOME EDITING IN ZEBRAFISH (2018)
  • Author(s): Theeranukul, P., Fukuyo, T., Klangnurak, W., Tokumoto, T.
  • Organizer: 日本動物学会第89回大会
• [Presentation] ゼブラフィッシュのstarmaker(stm)遺伝子のゲノム編集による機能解析 (2018)
  • Author(s): ワンラダクラングヌラック、福與健人、エムディレザヌッジャマン、徳元俊伸
  • Organizer: 日本動物学会第89回大会
• [Presentation] プロゲスチン膜受容体 (mPR) 遺伝子群変異メダカ系統の表現型解析 (2018)
  • Author(s): 王軍、Abdullah AN Naser 、徳元俊伸
  • Organizer: 第４３回日本比較内分泌学会大会
• [Presentation] ゼブラフィシュ生体内卵成熟・排卵誘導法による排卵誘導候補遺伝子の特異的選択 (2017)
  • Author(s): ワンラダクラングヌラック、福與健人、エムディレザヌッジャマン、今村聖実、堀内映実、菅野純夫、鈴木穣、徳元俊伸
  • Organizer: 日本動物学会第88回大会
• [Presentation] キンギョ脳におけるプロゲスチン膜受容体 (mPR) の局在 (2017)
  • Author(s): 王 軍、原口省吾、徳元俊伸
  • Organizer: 第４２回日本比較内分泌学会大会
• [Presentation] キンギョ脳におけるプロゲスチン膜受容体 (mPR) の発現とステロイドとの結合親和性 (2016)
  • Author(s): 王 軍、原口省吾、徳元俊伸
  • Organizer: 第４１回日本比較内分泌学会大会
  • Place of Presentation: 北里大学海洋生命科学部 神奈川県 相模原市
  • Year and Date: 2016-12-09
  • Int'l Joint Research
• [Presentation] An application of in vivo assay to identify ovulation-inducing genes in zebrafish (2016)
  • Author(s): Klangnurak W, Tosaka M, Imumura K, Horiuchi T, Sugano S, Suzuki Y, Tokumoto T.
  • Organizer: The joint meeting of the 22nd International Congress of Zoology and the 87th Meeting of the Zoological Society of Japan
  • Place of Presentation: 沖縄コンベンションセンター 沖縄県 那覇市
  • Year and Date: 2016-11-14
  • Int'l Joint Research
• [Presentation] Establishment of membrane progestin receptor (mPR) mutant Medaka strains to analyse central roles of mPRs (2016)
  • Author(s): J. Wang, S.R. Roy, I. Hara, K.Naruse, Y. Kamei, Y. Taniguchi, T. Tokumoto
  • Organizer: The joint meeting of the 22nd International Congress of Zoology and the 87th Meeting of the Zoological Society of Japan
  • Place of Presentation: 沖縄コンベンションセンター 沖縄県 那覇市
  • Year and Date: 2016-11-14
  • Int'l Joint Research
• [Presentation] Identification of ovulation-inducing genes selected by the method for in vivo induction of oocyte maturation and ovulation in zebrafish. (2016)
  • Author(s): Klangnurak W, Tosaka M, Imumura K, Horiuchi T, Sugano S, Suzuki Y, Tokumoto
  • Organizer: The Japanese Medaka and Zebrafish Meeting (JMZM)
  • Place of Presentation: 基礎生物学研究所 愛知県 岡崎市
  • Year and Date: 2016-08-20
  • Int'l Joint Research
• [Presentation] Approaches for detailed analysis of membrane progestin receptor (mPR) protein and for physiological roles of mPR (2016)
  • Author(s): T. Tokumoto
  • Organizer: 8th International Symposium of Fish Endocrinology
  • Place of Presentation: Lindholmen Conference Centre Gothenburg Sweden
  • Year and Date: 2016-06-28
  • Int'l Joint Research / Invited
• [Book] ナノバイオ・テクノロジー (2016)
  • Author(s): 徳元俊伸
  • Total Pages: 267
  • Publisher: 静岡学術出版