Tokmakov AA and Sato KI (2021), Modulation of Intracellular ROS and Senescence-...

XB-ART-58323

Antioxidants (Basel) 2021 Jul 01;107:. doi: 10.3390/antiox10071068.

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Modulation of Intracellular ROS and Senescence-Associated Phenotypes of Xenopus Oocytes and Eggs by Selective Antioxidants.

Tokmakov AA , Sato KI .

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Aging of oocytes and eggs diminishes their reproductive and developmental potential. It has been demonstrated previously that reactive oxygen species (ROS) contribute to accelerated aging of various cells. In the present study, we measured intracellular levels of ROS and investigated effects of several selective antioxidants (AOXs) on the viability and functional activity of aging oocytes and eggs of the African clawed frog Xenopus laevis. The fluorescent cell-permeable dye DCFDA, which is widely employed for ROS detection in cultured mammalian cells, was used to monitor ROS levels in the fresh and bench-aged oocytes and eggs by an optimized protocol. It was found that intracellular ROS contents were increased in frog oocytes and eggs aged for 48 h. It was further demonstrated using selective cell-permeable AOXs targeting different ROS-generating mechanisms, that the major source of ROS in Xenopus oocytes and eggs is the plasma membrane NADPH oxidase, and that mitochondrial generation contributes to the intracellular ROS content to a lesser extent. Targeted inhibition of NADPH oxidase with a natural organic compound apocynin reduced ROS levels significantly in Xenopus oocytes and eggs, maintained their normal phenotype and supported their functional competence. To our knowledge this is the first report concerning beneficial effects of apocynin on the isolated gamete cells, such as oocytes and eggs.

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Species referenced: Xenopus laevis
GO keywords: peroxidase activity [+]

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	Figure 1 Conversion of reactive oxygen species (ROS) and selectivity of applied antioxidants (AOXs). Panel (A) shows the pathways of intracellular conversion of ROS. Panels (B,C) describe functions and chemical structures, respectively, of the selective AOXs examined in this study.
	Figure 2 Optimization of ROS detection in Xenopus oocytes. In panel (A), the oocytes were incubated in the presence of 20 Î¼M DCFDA for the indicated times (0â€“120 min). At the end of incubation, the indicator was washed off and oocyte fluorescence was observed with the filters used for detection of GFP fluorescence. In panel (B), DCFDA was uploaded for 30 min, the drug was washed off, and the oocytes were further incubated in its absence over the oxidation period (0â€“18 h). Panel (C) shows fluorescent images of the oocytes that were analyzed in panel (B).
	Figure 2 Optimization of ROS detection in Xenopus oocytes. In panel (A), the oocytes were incubated in the presence of 20 Î¼M DCFDA for the indicated times (0â€“120 min). At the end of incubation, the indicator was washed off and oocyte fluorescence was observed with the filters used for detection of GFP fluorescence. In panel (B), DCFDA was uploaded for 30 min, the drug was washed off, and the oocytes were further incubated in its absence over the oxidation perioFigure 3 Figure 3 Detection of intracellular ROS in Xenopus oocytes and eggs with DCFDA. Oocytes and eggs were preincubated in the presence of 20 Î¼M DCFDA for 1 h followed by incubation in the absence of the dye for 2 more hours. Fluorescence of DCFDA was observed with the filters used for detection of GFP fluorescence. Panel (A) demonstrates correlation of DCFDA fluorescence with the intracellular level of hydrogen peroxide. Panel (B) shows optical and fluorescent images of oocytes and eggs aged on bench for 48 h, and quantification of DCFDA fluorescence is presented in panel (C). Asterisks in panel (C) indicate statistical difference from the control (p < 0.05).
	Figure 4 The effect of selective AOXs on ROS levels in oocytes and eggs. Defolliculated Xenopus oocytes (A) and freshly ovulated dejellied eggs (B) were treated with the indicated AOXs, at the concentrations specified in Section 2 âMaterials and Methodsâ for 2 h, then DCFDA fluorescence was detected as described in the legend to Figure 2. Asterisks indicate statistical difference from the control (p < 0.05).
	Figure 5 Effect of selective AOXs on the morphology of in vitro aged defolliculated Xenopus oocytes. Optical images of normal, mottling and dead oocytes are presented in panel (A), proportion of these cell phenotypes in aging oocyte populations treated with the indicated AOXs for 96 h is shown in panel (B). Panel (C) shows percentage of normal oocytes in the oocyte populations treated with the indicated concentrations of apocynin for 72 h, and panel (D) presents occurrence of germinal vesicle breakdown (GVBD) in the aged PG-treated oocytes, as estimated within 12 h of hormone administration. In panel (D), apocynin was extensively washed out for 1 h before PG addition to oocytes.
	Figure 7 Effect of apocynin on Xenopus egg fertilization. In panel (A), the eggs obtained from hormone-injected female frogs were aged in the presence or absence of apocynin over 24 h, counting from the time of egg deposition, then the AOX drug was extensively washed off for 1 h, and both treated and untreated eggs were fertilized at the same time. In panels (B,C), ovulated eggs were fertilized in the presence of apocynin at the indicated concentrations within 1 h after deposition.

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