Declining male fertility with age and the molecular mechanism to suppress age-dependent male fertility

• Project/Area Number: 19K09793
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 56040:Obstetrics and gynecology-related
• Research Institution: National Center for Child Health and Development
• Principal Investigator: Kang Woojin 国立研究開発法人国立成育医療研究センター, 細胞医療研究部, 研究員 (10647978)
• Co-Investigator(Kenkyū-buntansha): 河野 菜摘子 明治大学, 農学部, 専任准教授 (00451691) ; 宮戸 健二 国立研究開発法人国立成育医療研究センター, 細胞医療研究部, 室長 (60324844)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2020: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: クエン酸合成酵素 / カルシウムオシレーション / 妊孕性 / 非ミトコンドリア型 / eTCA回路 / 加齢 / 男性不妊

Outline of Research at the Start

TCA回路の律速酵素は『クエン酸合成酵素』であるが、この酵素には非ミトコンドリア型が存在することは知られていない。先行研究から、非ミトコンドリア型クエン酸合成酵素（extra-mitochondrial CS、以下eCS）もまた重要な役割を担っていることがわかってきた。eCSは精子に発現しており、卵内に入ることでカルシウム波を誘導し、eCs欠損精子は、カルシウム波の開始が非常に遅れることを明らかにした。さらに、eCs欠損雄マウスは、生後6か月（我々の約30歳に相当）で生殖能をほぼ失った。本研究では、eCSによる卵活性化機構を解明するとともに、ミトコンドリア外でのTCA回路の存在を明らかにする。

Outline of Final Research Achievements

One half of infertile couples have male factor causes such as azoospermia and oligozoospermia. However, the mechanism of declining male fertility with age is not well known. In this study, we focused on citrate synthase (CS), which is a core enzyme of the mitochondrial tricarboxylic acid (TCA) cycle, particular, extra-mitochondrial CS (eCS) abundant in the sperm head. We investigated the role of eCS in male fertility by producing eCs-deficient (eCs-KO) mice. The initiation of the first spike of Ca2+ oscillation was substantially delayed in eggs fused with eCs-KO sperm, despite normal expression of sperm factor phospholipase C zeta 1. The eCs-KO male mice were initially fertile, but the fertility dropped at 6 months of age (corresponded to about 30 years in human). The data suggest that eCS suppresses age-dependent male infertility, providing insights into the elucidation of the causes of various diseases including the decline of male fertility with age.

Academic Significance and Societal Importance of the Research Achievements

卵活性化機構について研究されているものの、候補因子の数が増えているだけで、研究の大きな進展はなかった。これまで「卵活性化」の精子ファクターはホスファリパーゼCゼータ1（PLCz1）、1つだと考えられていたが「クエン酸合成酵素」も精子ファクターとしての働くことを発見した。さらに、第2の精子ファクターである「クエン酸合成酵素」が加齢によりその働きが弱まり、精子内で合成される「クエン酸」含有量も減少すると男性不妊を発症する可能性があることから、男性不妊の分子的基盤の理解に貢献できる。今後、精子に含まれる「クエン酸」の含有量を測定することで男性不妊のリスク要因を評価する方法の開発などが期待される。

Research Products

• [Journal Article] Suppressive role of lactoferrin in overweight-related female fertility problems. (2022)
  • Author(s): Sato B, Kanai S, Sakaguchi D, Yajima K, Matsumoto Y, Morohoshi K, Kagaya S, Izumo N, Ichinose M, Kang W, Miyado M, Miyado K and Kawano N.
  • Journal Title: Nutrients Volume: 14 Issue: 5 Pages: 938-938
  • DOI: 10.3390/nu14050938
  • Peer Reviewed / Open Access
• [Journal Article] Cryopreservation of undifferentiated and differentiated human neuronal cells (2022)
  • Author(s): Yamatoya Kenji、Nagai Yuya、Teramoto Naozumi、Kang Woojin、Miyado Kenji、Nakata Kazuya、Yagi Tohru、Miyamoto Yoshitaka
  • Journal Title: Regenerative Therapy Volume: 19 Pages: 58-68
  • DOI: 10.1016/j.reth.2021.12.007
  • Peer Reviewed / Open Access
• [Journal Article] Emerging Role of TCA Cycle-Related Enzymes in Human Diseases (2021)
  • Author(s): Kang Woojin、Suzuki Miki、Saito Takako、Miyado Kenji
  • Journal Title: International Journal of Molecular Sciences Volume: 22 Issue: 23 Pages: 13057-13057
  • DOI: 10.3390/ijms222313057
  • Peer Reviewed / Open Access
• [Journal Article] Similar responsiveness between C57BL/6N and C57BL/6J mouse substrains to superovulation (2021)
  • Author(s): Miyuki Shindo, Hideki Tsumura, Kenji Miyado, Woojin Kang, Natsuko Kawano, Tomoko Yoshida, Maki Fukami, Mami Miyado
  • Journal Title: microPublication Biology Volume: 375 Pages: 375-375
  • Peer Reviewed / Open Access
• [Journal Article] Identification of an antibacterial polypeptide in mouse seminal vesicle secretions (2021)
  • Author(s): Morohoshi Kazunori、Yamazaki Takeo、Kito Keiji、Sato Ban、Kang Woojin、Hibino Taku、Yoshida Manabu、Yoshida Kaoru、Iwamoto Teruaki、Yamada Mitsutoshi、Miyado Kenji、Kawano Natsuko
  • Journal Title: Journal of Reproductive Immunology Volume: 148 Pages: 103436-103436
  • DOI: 10.1016/j.jri.2021.103436
  • Peer Reviewed / Open Access
• [Journal Article] Next-Generation Sequencing Reveals Downregulation of the Wnt Signaling Pathway in Human Dysmature Cumulus Cells as a Hallmark for Evaluating Oocyte Quality (2020)
  • Author(s): Ryosuke Akino, Daisuke Matsui, Ryouka Kawahara-Miki, Mitsuyoshi Amita, Kuniko Tatsumi, Eri Ishida, Woojin Kang, Shuji Takada, Kenji Miyado, Akihiko Sekizawa,Takakazu Saito,Tomohiro Kono, Hidekazu Saito
  • Journal Title: Reproductive Medicine Volume: 1 Issue: 3 Pages: 205-215
  • DOI: 10.3390/reprodmed1030016
  • Peer Reviewed / Open Access
• [Journal Article] Suppression of Non-Random Fertilization by MHC Class I Antigens (2020)
  • Author(s): Kamiya Junki、Kang Woojin、Yoshida Keiichi、Takagi Ryota、Kanai Seiya、Hanai Maito、Nakamura Akihiro、Yamada Mitsutoshi、Miyamoto Yoshitaka、Miyado Mami、Kuroki Yoko、Hayashi Yoshiki、Umezawa Akihiro、Kawano Natsuko、Miyado Kenji
  • Journal Title: International Journal of Molecular Sciences Volume: 21 Issue: 22 Pages: 8731-8731
  • DOI: 10.3390/ijms21228731
  • Peer Reviewed / Open Access
• [Journal Article] Neuronal expression of Ca 2+ oscillation initiator is linked to rapid neonatal growth in mice (2020)
  • Author(s): Woojin Kang, Kenji Yamatoya, Kenji Miyado, Mami Miyado, Yoshitaka Miyamoto
  • Journal Title: microPublication Biology Volume: 000325 Pages: 000325-000325
  • Peer Reviewed / Open Access
• [Journal Article] Human Semenogelin 1 Promotes Sperm Survival in the Mouse Female Reproductive Tract (2020)
  • Author(s): Sakaguchi Daiki、Miyado Kenji、Iwamoto Teruaki、Okada Hiroshi、Yoshida Kaoru、Kang Woojin、Suzuki Miki、Yoshida Manabu、Kawano Natsuko
  • Journal Title: International Journal of Molecular Sciences Volume: 21 Issue: 11 Pages: 3961-3961
  • DOI: 10.3390/ijms21113961
  • Peer Reviewed / Open Access
• [Journal Article] Extra-mitochondrial citrate synthase initiates calcium oscillation and suppresses age-dependent sperm dysfunction (2020)
  • Author(s): Kang Woojin、Harada Yuichirou、Yamatoya Kenji、Kawano Natsuko、Kanai Seiya、Miyamoto Yoshitaka、Nakamura Akihiro、Miyado Mami、Hayashi Yoshiki、Kuroki Yoko、Saito Hidekazu、Iwao Yasuhiro、Umezawa Akihiro、Miyado Kenji
  • Journal Title: Laboratory Investigation Volume: 100 Issue: 4 Pages: 583-595
  • DOI: 10.1038/s41374-019-0353-3
  • Peer Reviewed / Open Access
• [Journal Article] Microexosomes versus exosomes: Shared components but distinct structures (2019)
  • Author(s): Miyado Mami、Kang Woojin、Kawano Natsuko、Miyado Kenji
  • Journal Title: Regenerative Therapy Volume: 11 Pages: 31-33
  • DOI: 10.1016/j.reth.2019.04.013
  • Peer Reviewed / Open Access
• [Journal Article] Deletion of a Seminal Gene Cluster Reinforces a Crucial Role of SVS2 in Male Fertility (2019)
  • Author(s): Shindo Miyuki、Inui Masafumi、Kang Woojin、Tamano Moe、Tingwei Cai、Takada Shuji、Hibino Taku、Yoshida Manabu、Yoshida Kaoru、Okada Hiroshi、Iwamoto Teruaki、Miyado Kenji、Kawano Natsuko
  • Journal Title: International Journal of Molecular Sciences Volume: 20 Issue: 18 Pages: 4557-4557
  • DOI: 10.3390/ijms20184557
  • Peer Reviewed / Open Access
• [Journal Article] Membrane protein CD9 is repositioned and released to enhance uterine function. (2019)
  • Author(s): Iwai M, Hamatani T, Nakamura A, Kawano N, Kanai S, Kang W, Yoshii N, Odawara Y, Yamada M, Miyamoto Y, Saito T, Saito H, Miyado M, Umezawa A, Miyado K, Tanaka M.
  • Journal Title: Laboratory Investigation Volume: 99 Issue: 2 Pages: 200-209
  • DOI: 10.1038/s41374-018-0145-1
  • Peer Reviewed / Open Access
• [Presentation] 「卵活性化」の新たな精子ファクターを発見：「精子老化」にはクエン酸が関与する (2020)
  • Author(s): 康 宇鎭, 宮戸 健二
  • Organizer: 第20回日本生殖工学会シンポジウム
  • Invited
• [Presentation] 毛色によって男性の生殖能は判別できるのか？ (2019)
  • Author(s): 康 宇鎭, 大和屋 健二, 原田 裕一郎, 河野 菜摘子, 宮戸 健二
  • Organizer: 第 42 回日本分子生物学会年会
• [Remarks] 世界初・「卵活性化」の新たな精子ファクターを発見！
  • URL: https://www.ncchd.go.jp/press/2020/pr_20200115.html