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

31 年

4月30 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 30 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30 年

4月

01 日～平成31 年

3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:--Detection of SA-β-gal activity in ageing Xenopus eggs and oocytes;
Studies of subcellular localization of SA-β-gal in the oocytes and eggs;
Fluorescent detection of Cai2+. Monitoring Cai2+ in ageing Xenopus eggs and egg extracts;
Monitoring ATP levels in ageing and overactivated Xenopus eggs;
Evaluation of lysosomal acidification in ageing eggs by a fluorescent method;
Effect of calcium chelators and protein kinase inhibitors on postovulatory ageing of Xenopus oocytes and eggs.

７．共同利用研究の成果
During this year, we conducted biochemical and microscopic analyses concerning localization ofsenescence-associated β-galactosidase (SA-β-gal) in Xenopus oocytes and eggs. SA-β-gal activity was found to belocated mainly within a fraction of dense cytoplasmic granules that were extensively stained with thelysosome-specific dye LysoTracker. The granules were abundant in the cytoplasm and they had an average size of
8.9+5.6 µm. These properties allowed us to identify the SA-β-gal-containing granules as a subpopulation of yolkplatelets, specialized late endosomes that accumulate and store processed vitellogenin in frog oocytes. Further
analysis revealed an increase of SA-β-gal activity in Xenopus eggs, but not oocytes, aged in vitro over 48 hours. Ourresults suggest that acidification of the endosomal compartment during egg aging is responsible for this increase. In
the course of this study, an optimized method has been developed for detection of SA-β-gal in Xenopus oocytes andeggs, using the recently developed cell-permeable fluorescent substrate of β-galactosidase, SPiDER-β-gal.
We also studied effects of the calcium chelators, such as EGTA, BAPTA, BAPTA-AM, on hydrogen
peroxide-induced egg overactivation. Protein and energy homeostases were investigated in the overactivted eggs
based on measurements of intracellular ATP and cytosolic protein contents. Time-dependent lipofuscin accumulation
was observed in the hydrogen peroxide-treated Xenopus eggs.

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, Sato KI. (2019) Reconstitution of extracellular calcium signaling in Xenopus egg extracts. Methods
Mol Biol 1920:41-57.
Sato KI, Tokmakov AA. (2019) Membrane Microdomains as Platform to Study Membrane-Associated Events DuringOogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis. Methods Mol Biol 1920:59-73.
Tokmakov A.A., Sato KI. (2019) Activity and intracellular localization of senescence-associated b-galactosidase inaging Xenopus oocytes and eggs. Exp Gerontology 119:157-167.
Tokmakov AA, Sato KI. (2018) Detection of SA-β-gal activity in Xenopus oocytes using SPiDER-β-gal. The 41st
Annual Meeting of the Molecular Biological Society of Japan, November 30th, 2018, Yokohama, Japan.
Isobe T., Sato K., Yoshida H., Awamura M., Hokatsu I., Matsumoto Y., Sato K-I., Tokmakov A. (2018) In vitro
reconstruction of oocyte life cycle: from ovulation to apoptosis. November 30th, 2018, Yokohama, Japan.

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。