様式３
神戸大学バイオシグナル総合研究センター共同利用研究報告書
平成神戸大学バイオシグナル総合研究センター長

30 年

04 月

16 日

殿所属機関・部局名
職名
研 究 代 表 者 名

京都産業大学 総合生命科学部
研究員
Tokmakov Alexander A.

下記のとおり平成２９年度の共同利用研究成果を報告します。
記（課題番号： 281027）
１．共同利用研究課題名

２．共同利用研究目的

Postovulatory ageing of Xenopus eggs

The study is aimed at the investigation of intracellular processes that occur upon ageing ofXenopus frog eggs. The focus is set on the mechanisms of spontaneous activation and meiotic
exit in mature metaphase-arrested eggs awaiting fertilization.

３．共同利用研究期間

平成29 年

04 月

01 日～平成30 年

03 月

31 日

４．共同利用研究組織
氏名(研究代表者)
Tokmakov A.A.

所属部局等

職名等

役 割 分 担京都産業大学
総合生命科学部

研究員

80%

摂南大学 理工学部生命科学科

講師

20%

(分担研究者)
Ijiri Takashi W.

５．センター内受入研究者

研究部門・分野名

Signal Function ・ Cell
proliferation
anddifferentiation

氏 名

Iwasaki Tetsushi※ 次の６，７，８の項目は，枠幅を自由に変更できます。但し，６，７，８の項目全体では１頁に収めて下さい。

（課題番号：281027）
６．共同利用研究計画
The following tasks were pursued during this year of study:- Fluorescent detection of (Cai2+). Monitoring (Cai2+) in ageing, activated and hyperactivated eggs;- Detection of calpain and cathepsin activity in ageing, activated, and hyperactivated eggs;- Monitoring of ATP levels in ageing Xenopus eggs;- Evaluation of the mitochondrial membrane potential by a fluorescent method, monitoring mitochondrial
membrane potential in ageing Xenopus eggs;- Detection of intracellular ROS, monitoring ROS levels in ageing Xenopus eggs;- Effect of calcium chelators, protein kinase inhibitors and ROS scavengers on postovulatory ageing.

７．共同利用研究の成果
A fluorescent method of Cai2+ detection in Xenopus oocytes and eggs based on the use of Fluo-8 indicator wasemployed to monitor levels of intracellular Cai2+ in resting oocytes and eggs, and the cells activated or hyperactivatedwith H2O2. Various patterns of calcium signal were observed in these models. It was found that egg hyperactivation
led to a prolonged uncompensated release of intracellular calcium, while only a transient calcium signal wasobserved in the activated eggs.
In accordance with the kinetics of Cai2+ release, the sequential activation of calpain and cathepsin was detected in theactivated and hyperactivated eggs.
Monitoring of ROS levels in Xenopus eggs and oocytes treated with selective antioxidants (AOX) revealed that thespecific inhibitors of the membrane NADPH-oxidase can potently modulate intracellular ROS levels and stabilize
oocytes and eggs. Dose-dependent effects of 4-hydroxy-3-methoxyactophenone (apocynin) were further investigatedin detail.
The inhibitory effect of the MAPK pathway inhibitor U0126 on the timing of maturation, ovulation, and apoptotic
initiation was revealed.

８．共同利用研究成果の学会発表・研究論文発表状況（本センターの担当教員の氏名の記載，又はこの共同利用研究に基づくとの記載のある論文等を記載して下さい。
なお，論文の場合は，別刷りを１部提出してください。）
Tokmakov AA, Iguchi S, Iwasaki T, Sato KI. (2017) Activity and distribution of senescence associatedbeta-galactosidase in aging oocytes and eggs. The 42nd FEBS Congress, September 13th,2017, Jerusalem, Israel.
Tokmakov AA, Sato K. (2017) Effect of selective antioxidants on stability Xenopus oocytes and eggs. 88th Annual
Meeting of the Zoological Society of Japan, September 21st, 2017, Toyama, Japan.
Ijiri TW, Shiba K, Sato H, Kawano N. (2018) Organizers of 第6_回生殖若手の会 筑波大学下田臨海実験センター
February 28th - March 2nd, 2018, Shimoda, Japan.
Tokmakov AA, Sato KI, Stefanov VE. (2018) Postovulatory cell death: why eggs die by apoptosis in biologicalspecies with external fertilization. J Reprod Dev 64(1): 1-6.

９．共同利用研究に関連した受賞，博士学位論文の取得，大型研究プロジェクトや競争的資金の獲得等がありましたらご記入ください。