Isolation and functional analysis of genes controlling totipotency

2020 Fiscal Year Annual Research Report

• Project Area: Program of totipotency: From decoding to designing
• Project/Area Number: 19H05750
• Research Institution: Osaka University
• Principal Investigator: 伊川 正人 大阪大学, 微生物病研究所, 教授 (20304066)
• Co-Investigator(Kenkyū-buntansha): 篠原 隆司 京都大学, 医学研究科, 教授 (30322770)
• Project Period (FY): 2019-06-28 – 2024-03-31
• Keywords: ゲノム編集 / 精子成熟 / 受精 / 卵活性化 / 全能性 / 不妊

Outline of Annual Research Achievements

本課題では、①生命起動メカニズムの解明、②卵活性化機構の解明をメインテーマとし、さらに③精子エピゲノム獲得機構の解明をサブテーマ として研究を進めている。
①生命起動メカニズムの解明：2020年度も引き続き、卵・卵巣特異的に発現する遺伝子について、CRISPR/Cas9 によりKOマウスを作製し、交配テストを実施した。その結果、PABPN1Lを欠損した卵では、GV期からMII期にかけて母性mRNAの分解が上手くいかず、受精後の発生が2細胞期に停止することを明らかにした(論文投稿準備中)。さらに、これら分解されるべきはずのmRNAについて着目しRNAseq解析を実施し、ZAR1L因子を見つけた。ホモログであるZAR1をKOすると初期胚発生停止することから、ZAR1LのKOマウスを作製したところ、残念ながら雌の妊孕性に異常は認められなかった。現在は、他因子のKOマウスを作製して解析中である。
②卵活性化機構の解明：受精卵は受精刺激により活性化される。PLCZ1 以外に受精・活性化に影響する因子を探索して機能解析している中で、卵との融合に必須な因子としてFIMP, SOF1, TMEM95, SPACA6の4因子を新たに同定して機能解析を行い、論文報告した。
③精子エピゲノム獲得機構の解明：分担者の篠原と協力して、精巣での精子形成から成熟に関わる因子を標的に受精・全能性発揮に必要な因子の探索を開始した。精巣由来で精巣上体の成熟を促すルミクリン現象に着目し、精巣で作られるNELL2がルミクリン因子として働き、受容体であるROS1が発現する精巣上体の上皮細胞の分化を促し、さらに分化した上皮細胞から分泌されるOVCH2プロテアーゼが精子ADAM3のトリミングを介して、精子受精能力と雄妊孕性に必須であることを明らかにした。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

卵特異的に発現する遺伝子群についてKOマウスの作製と解析は順調に進んでいる。すでに5つの因子が必須でないことを明らかにすると同時に、必須因子もいくつか同定しており、2021年度以降は解析に重点を移す。そのほか、40年来の謎であったルミクリンの機構を明らかにしてScience誌に報告、また精子成熟・受精因子をPNAS誌に3報報告するなど、順調に進んでいる。

Strategy for Future Research Activity

引き続き、篠原の得意とするGS細胞、伊川が得意とするゲノム編集のメリットを活用し、受精および卵活性化・受精後発生に寄与する因子の同定と機能解析を進める。しかしながら、コロナウイルス感染症対策もあり、動物飼育の見通しも不透明であるため、試験管培養を用いたアプローチなど代替策を検討する。

Research Products

• [Int'l Joint Research] Baylor College of Medecine/University of Hawai(米国)
  • Country Name: U.S.A.
  • Counterpart Institution: Baylor College of Medecine/University of Hawai
• [Int'l Joint Research] Seoul National University(韓国)
  • Country Name: KOREA (REP. OF KOREA)
  • Counterpart Institution: Seoul National University
• [Int'l Joint Research] Fudan University(中国)
  • Country Name: CHINA
  • Counterpart Institution: Fudan University
• [Journal Article] ARMC12 regulates spatiotemporal mitochondrial dynamics during spermiogenesis and is required for male fertility. (2021)
  • Author(s): Shimada K, Park S, Miyata H, Yu Z, Morohoshi A, Oura S, Matzuk MM, Ikawa M.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 118 Pages: e2018355118
  • DOI: 10.1073/pnas.2018355118.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Baek SH. Protocol for isolation of spermatids from mouse testes. (2021)
  • Author(s): Kim CR, Noda T, Okada Y, Ikawa M.
  • Journal Title: STAR Protoc. Volume: 2 Pages: 100254
  • DOI: 10.1016/j.xpro.2020.100254.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia (2021)
  • Author(s): Morimoto H, Yamamoto T, Miyazaki T, Ogonuki N, Ogura A, Tanaka T, Kanatsu-Shinohara M, Yabe-Nishimura C, Zhang H, Pommier Y, Trumpp A, Shinohara T.
  • Journal Title: Genes Dev . Volume: 35 Pages: 250-260
  • DOI: 10.1101/gad.339903.120
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] OGG1 protects mouse spermatogonial stem cells from reactive oxygen species in culture (2021)
  • Author(s): Mori Y, Ogonuki N, Hasegawa A, Kanatsu-Shinohara M, Ogura A, Wang Y, McCarrey JR, Shinohara T.
  • Journal Title: Biol Reprod . Volume: 104 Pages: 706-716
  • DOI: 10.1093/biolre/ioaa216
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] CRISPR/CAS9-mediated amino acid substitution reveals phosphorylation residues of RSPH6A are not essential for male fertility in mice†. (2020)
  • Author(s): Miyata H, Abbasi F, Visconti PE, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 912-914
  • DOI: 10.1093/biolre/ioaa161.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] PHF7 Modulates BRDT Stability and Histone-to-Protamine Exchange during Spermiogenesis. (2020)
  • Author(s): Kim CR, Noda T, Kim H, Kim G, Park S, Na Y, Oura S, Shimada K, Bang I, Ahn JY, Kim YR, Oh SK, Choi HJ, Kim JS, Jung I, Lee H, Okada Y, Ikawa M, Baek SH.
  • Journal Title: Cell Rep. Volume: 32 Pages: 107950
  • DOI: 10.1016/j.celrep.2020.107950.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Bi-allelic DNAH8 Variants Lead to Multiple Morphological Abnormalities of the Sperm Flagella and Primary Male Infertility. (2020)
  • Author(s): Liu C, Miyata H, Gao Y, Sha Y, Tang S, Xu Z, Whitfield M, Patrat C, Wu H, Dulioust E, Tian S, Shimada K, Cong J, Noda T, Li H, Morohoshi A, Cazin C, Kherraf ZE, Arnoult C, Jin L, He X, Ray PF, Cao Y, Touré A, Zhang F, Ikawa M.
  • Journal Title: Am J Hum Genet. Volume: 107 Pages: 330-341
  • DOI: 10.1016/j.ajhg.2020.06.004.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Knockout of family with sequence similarity 170 member A (Fam170a) causes male subfertility, while Fam170b is dispensable in mice†. (2020)
  • Author(s): Devlin DJ, Nozawa K, Ikawa M, *Matzuk MM.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 205-222
  • DOI: 10.1093/biolre/ioaa082.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CRISPR/Cas9-based genome editing in mice uncovers 13 testis- or epididymis-enriched genes individually dispensable for male reproduction†. (2020)
  • Author(s): Sun J, Lu Y, Nozawa K, Xu Z, Morohoshi A, Castaneda JM, Noda T, Miyata H, Abbasi F, Shawki HH, Takahashi S, Devlin DJ, Yu Z, Matzuk RM, Garcia TX, Matzuk MM, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 183-194
  • DOI: 10.1093/biolre/ioaa083.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity. (2020)
  • Author(s): Park S, Shimada K, Fujihara Y, Xu Z, Shimada K, Larasati T, Pratiwi P, Matzuk RM, Devlin DJ, Yu Z, Garcia TX, Matzuk MM, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 195-204
  • DOI: 10.1093/biolre/ioaa084.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Tmprss12 is required for sperm motility and uterotubal junction migration in mice†. (2020)
  • Author(s): Larasati T, Noda T, Fujihara Y, Shimada K, Tobita T, Yu Z, Matzuk MM, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 254-263
  • DOI: 10.1093/biolre/ioaa060.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] NELL2-mediated lumicrine signaling through OVCH2 is required for male fertility. Science. (2020)
  • Author(s): Kiyozumi D, Noda T, Yamaguchi R, Tobita T, Matsumura T, Shimada K, Kodani M, Kohda T, Fujihara Y, Ozawa M, Yu Z, Miklossy G, Bohren KM, Horie M, Okabe M, Matzuk MM, Ikawa M.
  • Journal Title: Science. Volume: 368 Pages: 1132-1135
  • DOI: 10.1126/science.aay5134.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] CIB4 is essential for the haploid phase of spermatogenesis in mice†. (2020)
  • Author(s): Xu Z, Miyata H, Kaneda Y, Castaneda JM, Lu Y, Morohoshi A, Yu Z, Matzuk MM, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 235-243
  • DOI: 10.1093/biolre/ioaa059.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm-oocyte fusion in mice. (2020)
  • Author(s): Noda T, Lu Y, Fujihara Y, Oura S, Koyano T, Kobayashi S, Matzuk MM, Ikawa M.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 117 Pages: 11493-11502
  • DOI: 10.1073/pnas.1922650117
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes globozoospermia and sterility in male mice†. (2020)
  • Author(s): Nozawa K, Zhang Q, Miyata H, Devlin DJ, Yu Z, Oura S, Koyano T, Matsuyama M, Ikawa M, Matzuk MM.
  • Journal Title: Biol Reprod. Volume: 103 Pages: :244-253
  • DOI: 10.1093/biolre/ioaa040.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Prss55 but not Prss51 is required for male fertility in mice†. (2020)
  • Author(s): Kobayashi K, Endo T, Matsumura T, Lu Y, Yu Z, Matzuk MM, Ikawa M.
  • Journal Title: Biol Reprod. Volume: 103 Pages: 223-234
  • DOI: 10.1093/biolre/ioaa041.
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Spermatozoa lacking Fertilization Influencing Membrane Protein (FIMP) fail to fuse with oocytes in mice. (2020)
  • Author(s): Fujihara Y, Lu Y, Noda T, Oji A, Larasati T, Kojima-Kita K, Yu Z, Matzuk RM, Matzuk MM, Ikawa M.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 117 Pages: 9393-9400
  • DOI: 10.1073/pnas.1917060117
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Testis-enriched kinesin KIF9 is important for progressive motility in mouse spermatozoa. (2020)
  • Author(s): Miyata H, Shimada K, Morohoshi A, Oura S, Matsumura T, Xu Z, Oyama Y, Ikawa M.
  • Journal Title: FASEB J. Volume: 34 Pages: 5389-5400
  • DOI: 10.1096/fj.201902755R.
  • Peer Reviewed / Open Access
• [Journal Article] Autologous transplantation of spermatogonial stem cells restores fertility in congenitally infertile mice (2020)
  • Author(s): Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, Shinohara T.
  • Journal Title: Proc Natl Acad Sci U S A . Volume: 117 Pages: 7837-7844
  • DOI: 10.1073/pnas.1914963117
  • Peer Reviewed / Open Access
• [Journal Article] CD2 is a surface marker for mouse and rat spermatogonial stem cells (2020)
  • Author(s): Kanatsu-Shinohara M, Chen G, Morimoto H, Shinohara T.
  • Journal Title: J Reprod Dev. Volume: 66 Pages: 341-349
  • DOI: 10.1262/jrd.2020-019
  • Peer Reviewed / Open Access
• [Presentation] ゲノム編集と受精研究への応用 (2021)
  • Author(s): 伊川正人
  • Organizer: 第17回熊本大学生命資源・研究センターシンポジウム
  • Invited
• [Presentation] ルミクリンシステムによる精子成熟の制御メカニズム (2020)
  • Author(s): 伊川正人
  • Organizer: 配偶子インテグリティの構築』『全能性プログラム』合同公開シンポジウム2020
• [Presentation] CRISPR/Cas9 Mediated Genome Editing and Its Application for the Study of Fertilization in Mice (2020)
  • Author(s): Masahito Ikawa
  • Organizer: The University of Kansas Medical Center seminar
  • Int'l Joint Research / Invited
• [Presentation] ゲノム編集マウスが切り開く受精研究の最前線 (2020)
  • Author(s): 伊川正人
  • Organizer: 第38回日本受精着床学会総会・学術講演会
  • Invited
• [Presentation] ゲノム編集マウスが切り開く受精研究の最前線 (2020)
  • Author(s): 伊川正人
  • Organizer: 第113回日本繁殖生物学会大会
  • Invited
• [Remarks] 研究室ホームページ
  • URL: https://egr.biken.osaka-u.ac.jp/