Oxidative Stress-Induced Overactivation of Frog Eggs Triggers Calcium-Dependent Non-Apoptotic Cell Death

Abstract

Excessive activation of frog eggs (overactivation) is a pathological process that renders eggs unfertilizable. Its physiological inducers are unknown. Previously, oxidative stress was shown to cause time- and dose-dependent overactivation of Xenopus laevis frog eggs. Here, we demonstrate that the oxidative stress-induced egg overactivation is a calcium-dependent phenomenon which can be attenuated in the presence of the selective calcium chelator BAPTA. Degradation of cyclin B2, which is known to be initiated by calcium transient in fertilized or parthenogenetically activated eggs, can also be observed in the overactivated eggs. Decline in mitochondrial membrane potential, ATP depletion and termination of protein synthesis manifest in the eggs within one hour of triggering overactivation. These intracellular events occur in the absence of caspase activation. Furthermore, plasma membrane integrity is compromised in the overactivated eggs, as evidenced by ATP leakage and egg swelling. In sum, our data demonstrate that oxidative stress-induced overactivation of frog eggs causes fast and dramatic disruption of cellular homeostasis, resulting in robust and expedited cell death by a calcium-dependent non-apoptotic mechanism.

Keywords: Xenopus laevis; cell death; eggs; overactivation; oxidative stress.

Figure 1

Involvement of calcium in the oxidative stress-induced overactivation of frog eggs. The progression of morphological changes in the overactivated eggs is presented in panel A. At time “0”, a population of Xenopus eggs was treated with 10 mM hydrogen peroxide, and a responsive (i.e., overactivated) egg was monitored over one hour following drug administration (panel (A)). Phenotypes of overactivated eggs treated with hydrogen peroxide for 12 h in the presence or absence of the selective calcium chelator BAPTA-AM (100 μM) are shown in panel (B). The rates of overactivation in the presence or absence of BAPTA or BAPTA-AM at different times are shown in panel (C). The experiment was repeated with three separate batches of eggs obtained from different animals and the result of a single-batch experiment is presented.

Figure 2

Cyclin B degradation in overactivated frog eggs. Cyclin B contents in the overactivated Xenopus eggs incubated with hydrogen peroxide for different times is presented in panels (A,B). Cyclin B contents in metaphase II-arrested (E), overactivated (OA), apoptotic (Apo) eggs and oocytes (O) are presented in panels (C,D). Panels (B,D) show quantification of the blots displayed in panels (A,C). In panels (C,D), overactivated eggs were collected after 1 h peroxide treatment, and apoptotic eggs were analyzed within 24–30 h of ovulation. The experiment was repeated with three separate batches of eggs obtained from different animals and the result of a single-batch experiment is shown. Bars in panels (B,D) represent SD values of the mean obtained in four measurements of the same experiment. Asterisks in the panels indicate statistical difference (p < 0.05) from untreated control eggs (0 min in panel (B), and E in panel (C)).

Figure 3

Caspase activity in overactivated frog eggs. Caspase 3/7 activity in the overactivated Xenopus eggs incubated with hydrogen peroxide for different times is presented in panels (A,B). Caspase activity in metaphase II-arrested (E), overactivated (OA), apoptotic (Apo) eggs, and oocytes (O) is presented in panels (C,D). Spot assays of caspase activity are shown in panels (A,C) and their quantification is presented in panels (B,D). In panels (C,D), overactivated eggs were collected after 1 h peroxide treatment, whereas apoptotic eggs were analyzed within 24–30 h of ovulation. The experiment was repeated with three separate batches of eggs obtained from different animals and the result of a single-batch experiment is shown. Bars in panels (B,D) represent SD values of the mean obtained in four measurements of the same experiment. Asterisks in the panels indicate statistical difference (p < 0.05) from untreated control eggs (E).

Figure 4

Decrease of the mitochondrial membrane potential in overactivated and apoptotic eggs. Morphological types of metaphase II-arrested (E), overactivated (OA), apoptotic (Apo) eggs and oocytes (O) are presented in panel (A). Corresponding fluorescent images of intracellular compartments, stained with the MitoTracker, are shown in panel (B). Panel (C) shows quantification of the data presented in panel (B). Fluorescent signals from more than ten individual mitochondria were evaluated in each cell type. Asterisks in panel (C) denote statistical difference (p < 0.05) from untreated control eggs (E).

Figure 5

ATP content and protein synthesis in the frog eggs overactivated by oxidative stress. Xenopus oocytes were matured in vitro in the presence of progesterone and incubated in the presence or absence of hydrogen peroxide. Panel (A) shows the content of intracellular ATP in the overactivated eggs treated with 10 mM hydrogen peroxide for one hour and peroxide-untreated frog oocytes (PG−) and eggs (PG+), as evaluated by a chemiluminescent assay (see Section 2, “Materials and Methods” for details). Panel (B) presents the intensity of a luminescent signal generated by the luciferase protein synthesized in the oocytes and eggs after injection of luciferase mRNA. The luciferase reporter mRNA was microinjected into the cells immediately before hydrogen peroxide administration. Four to six oocytes or eggs were examined under each condition of treatment. Asterisks denote statistical difference (p < 0.05) from untreated control oocytes (PG−, H₂O₂−).

Figure 6

Breach of plasma membrane integrity in overactivated eggs. In vitro matured Xenopus eggs were treated with 10 mM hydrogen peroxide for one hour. Evaluations of intracellular (In) and extracellular (Ex) ATP in the cases of untreated (Control) and treated (OA 1 h) eggs are presented in panel (A). The progressive increase in the diameter of overactivated eggs is disclosed in panel (B). Panel (A) presents data of four to six ATP measurements in the same batch of eggs. More than ten eggs were analyzed in panel B at each time point. Asterisks denote statistical difference (p < 0.05) from untreated control eggs.

Figure 7

Oxidative stress-induced overactivation and deterioration of frog eggs. Strong oxidative stress triggers overactivation and cell death of mature metaphase II-arrested eggs of Xenopus laevis by a calcium-dependent non-apoptotic mechanism (see details in text).