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Biology of Reproduction 62, 1745-1753 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Involvement of Mitochondria in Oxidative Stress-Induced Cell Death in Mouse Zygotes1

Lin Liua, James R. Trimarchib, and David L. Keefe2,a

a Department of Obstetrics & Gynaecology, Women & Infants Hospital, Brown University, Providence, Rhode Island 02905 b Marine Biological Laboratory, Woods Hole, Massachusetts 02543


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 µM for 15 min) hydrogen peroxide (H2O2) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H2O2 induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress and mitochondrial decay are likely to be major contributors to aging and aging-associated cell death [1–7]. Hydrogen peroxide (H2O2), a form of reactive oxygen species (ROS), mediates mitochondrial oxidative damage that may lead to mitochondrial dysfunction and ultimately cell death, particularly when the mitochondrial antioxidant defense mechanisms are compromised, such as occurs during aging [8–11]. Alterations in mitochondrial structure and function have been shown to occur early during programmed cell death (PCD) or apoptosis, and mitochondria appear to be central regulators of apoptosis [12–15].

Oxidative stress, depending on its severity, can lead to either cell necrosis or apoptosis [16]. Necrosis is typified by cell and organelle swelling and leakage of intracellular contents into the extracellular milieu, resulting in inflammatory reaction. In contrast, apoptosis is characterized by cell shrinkage, membrane blebbing, and chromatin condensation [17–19]. However, the same inducer can cause either apoptosis or necrosis, depending on dose and duration [20]. Furthermore, secondary necrosis may occur during late stages of PCD [21] and may simply reflect an insufficient removal of apoptotic cells by phagocytes or neighboring cells [17]. Analyzing PCD in zygotes has advantages over other somatic cell systems in that zygotes are individual cells, and therefore confounding signaling from neighboring cells is absent. Moreover, effects of apoptotic signaling on subsequent development can be monitored in vitro.

Mitochondria mediate both apoptosis and necrotic forms of cell death [22]. The role of mitochondria in apoptosis of eggs is of interest because mitochondrial structure in eggs differs from that of somatic cell types. In eggs, mitochondria are electron-dense, with no obvious cristae from inner membrane [23], and therefore have been called immature. Zygotes can simply utilize pyruvate for energy production and produce limited ATP [24, 25]. It is unknown whether the immature mitochondria in oocytes or zygotes function differently during cell death, compared to mature mitochondria in most somatic cells.

Understanding PCD in mammalian embryos also may have clinical implications. Age-related decline in female fertility is a common phenomenon in older women and other long-lived mammals [26]. Maternal age has been demonstrated to affect oocyte quality and early embryonic development [27, 28]. During development, apoptotic cell death mediates follicular atresia and oocyte degeneration [29, 30]. Recently, it was found that PCD occurs in embryos that fail to execute essential developmental events during the first cell cycle [31]. In addition, both Bcl-2 (apoptotic inhibitor) and Bax (proapoptotic molecule) mRNA are present in preimplantation embryos [18, 32]. H2O2 has been shown to mediate PCD in the blastocyst [33], and fragmented human embryos contain high levels of H2O2 and exhibit evidence of PCD [34]. Elevated H2O2 levels also may be associated with the ``2-cell block" that occurs in some mouse strains [35]. While there are no laboratory animal models for naturally occurring PCD during normal early embryo development, pharmacological models of mild oxidative stress might be an alternative way to mimic the chronic low levels of oxidative stress associated with aging.

The present studies first tested the hypothesis that mild or intensive oxidation caused by H2O2 induces PCD in mammalian zygotes. As mild oxidative stress more closely mimics chronic accumulative oxidative stress that occurs during aging [36], we further examined changes in mitochondrial structure and function after exposure to mild oxidation treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents, Animals, Embryo Collection, and Culture

All reagents were purchased from Sigma Chemical Co. (St. Louis, MO), unless stated otherwise. Equine CG, used for superovulation, was purchased from Calbiochem (La Jolla, CA). B6C3F1 female mice, 6 wk old, were purchased from Charles River Laboratories (Boston, MA) and subjected to a 14L:10D light cycle for at least 1 wk before use. Animals were cared for according to procedures approved by the Marine Biological Laboratory and Women and Infants Hospital Animal Care Committees. Female mice were superovulated by i.p. injection of 7.5 IU eCG, followed 46–48 later by injection of 7.5 IU hCG, and then mated individually with B6C3F1 males with proven fertility. Next morning, females with vaginal plugs were selected and killed by cervical dislocation at 21–22 h after hCG injection. Zygotes (Day 1) enclosed in cumulus masses were released from the ampullae into KSOM (potassium simplex optimized medium) [37], buffered with 14 mM Hepes and 4 mM sodium bicarbonate, containing 0.03% hyaluronidase; then cumulus cells were removed by gentle pipetting [38]. Cumulus-free zygotes were washed and cultured in the KSOM supplemented with nonessential amino acids and 2.5 mM Hepes [37–39]. Embryos were cultured in 50-µl droplets of KSOM under mineral oil at 37°C in a humidified atmosphere of 7% CO2 in air. H2O2 were appropriately diluted in KSOM for treatment of zygotes. Embryos were pooled and randomly distributed to each treatment group. Embryos were assessed for cleavage at Day 2 and then development to blastocysts at Day 4. Based on dose-response experiments, appropriate concentrations of H2O2 were chosen for further analysis in cell death experiments.

Live and Dead Assay

For cell death assessment, both the cell-impermeant dye propidium iodide (PI) and the cell-permeant dye Hoechst and fluorescence microscopy were employed [40, 41]. Embryos were stained with 20 µg/ml PI (Molecular Probes, Eugene, OR) and the cell-permeant dye Hoechst 33342 (20 µg/ml) for 15 min, washed, and then observed under an inverted microscope (Zeiss Axiovert 100TV, Oberkochen, Germany) equipped with fluorescence optics. Viable zygotes displayed a normal nuclear size and blue fluorescence in pronuclei under UV illumination. Dead zygotes manifested PI-positive stain (red) in pronuclei and brighter blue fluorescence in the cytoplasm. In addition, blebs of plasma membrane, shrinkage of cells, and condensation or fragmentation of nuclei also were considered indicative of apoptosis [18]. These morphological changes still provide the most reliable criteria for recognizing apoptosis [19].

Alternatively, the live/dead viability/cytotoxicity kit (Molecular Probes) [42] was used to detect live and dead embryos. Embryos were incubated with 2 µM calcein AM and 4 µM ethidium homodimer solution at 37°C for 20 min. Fluorescence was detected using a Zeiss Axiovert 100TV inverted fluorescence microscope. Live embryos displayed intense uniform green fluorescence (fluorescein filter), whereas dead embryos showed no green fluorescence but rather bright red fluorescence (rhodamine filter) (see Fig. 1). The results obtained from this live/dead kit were consistent with those obtained with the PI and Hoechst stains.



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  		FIG. 1. Live and dead detection of cell death using the live/dead kit. Live zygotes exhibited green fluorescence throughout the cytoplasm under the fluorescein filter. Dead zygotes showed no cytoplasmic green fluorescence but strong red fluorescence, especially in the pronuclei. PN, Pronucleus; PB, polar body

Detection of Apoptosis by Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling (TUNEL) Technique

Embryos were fixed in 3.7% paraformaldehyde in Dulbecco's PBS containing 0.1% polyvinylpyrrolidone (PVP). Nuclear DNA fragmentation in embryos was detected by the TUNEL method using the in situ cell death detection kit (Boehringer-Mannheim, Indianapolis, IN) and nuclei were counterstained with PI (50 µg/ml; Molecular Probes), as described previously [43, 44]. Fluorescence was detected using a Zeiss LSM 510 laser scanning confocal microscope or Zeiss Axiovert 100TV inverted fluorescence microscope.

Immunocytochemistry for Localization of Cytochrome c

Release of cytochrome c from mitochondria was assessed by immunocytochemistry [44–46], with some minor modifications made for assessment in mouse embryos. Briefly, embryos were fixed overnight at 4°C in 4% paraformaldehyde and permeabilized in 0.3% Triton X-100 for 30 min at room temperature. After being washed, embryos were blocked with PBS-PVP, supplemented with 5% normal goat serum for 45 min, then incubated with a mouse monoclonal anti-cytochrome c antibody (PharMingen, San Diego, CA) diluted at 1:50 in PBS-PVP supplemented with 5% normal goat serum for 2 h at room temperature. Embryos were washed extensively with PBS-PVP, then incubated with Texas Red anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA) diluted 1:100 for 45 min. Nuclei were stained with Hoechst 33258 (20 µg/ml) for 15 min. Embryos were mounted on a glass slide in Vectashield mounting medium (H-1000, Vector) and sealed. Cytochrome c localization was detected by fluorescence microscopy using Texas Red filter (excitation 595 nm, emission 610–615 nm) and UV filter for nuclear stain.

Caspase Activation Assay

Embryos were fixed in 4% paraformaldehyde overnight at 4°C, then blocked for 30 min in blocking serum solution (PBS-PVP supplemented with 5% normal goat serum and 0.3% Triton X-100). Afterward, embryos were incubated with a polyclonal rabbit anti-active caspase-3 antibody (PharMingen) diluted at 1:100 in blocking serum solution overnight at 4°C. Embryos were washed thoroughly and then incubated in ABC solution with Vectastain Elit ABC Kit (Vector). Samples were reacted with diaminobenzidine solution until the desired brown intensity was obtained. The embryos were mounted on a slide and viewed with a transmitted light microscope.

Detection of Mitochondrial Membrane Potential (MMP) and Distribution of Active Mitochondria

MMP was measured with the lipophilic, cationic probe 5,5',6,6'-tetrachloro-1,1'3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) as described in previous reports [47, 48]. In the presence of a high MMP, JC-1 forms J-aggregates that emit red fluorescence, while JC-1 monomeric form emits green fluorescence at low MMP. Both colors were detected using a confocal microscope (Zeiss LSM510) with excitation at 488 nm and beam path control setting at LP 585 nm for Ch1 and BP 505–530 nm for Ch2. Embryos were incubated in 100 µl Hepes-buffered KSOM containing 1.25 µM JC-1 for 20 min at 37°C. Ratio analysis was performed with Zeiss LSM510 software and MetaMorph imaging software (UIC, Boston, MA).

The distribution of active mitochondria within cytoplasm was determined by rhodamine 123 (Rh123) [38, 49] or MitoTracker Red (personal communications with B.D. Bavister's laboratory, University of Wisconsin, Madison). Zygotes were incubated with either 10 µg/ml Rh123 or 330 nM MitoTracker Red (Molecular Probes) for 15 min, washed, and then observed with the same Zeiss fluorescence microscope as described above. Rh123 and MitoTracker showed the same staining pattern of active mitochondrial distribution, but the red fluorescence stained with MitoTracker lasted longer.

Transmission Electronic Microscopy

Embryos were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C for 1 h, washed in 0.1 M phosphate buffer, and embedded in agar chips. Subsequently, they were postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.2; they were dehydrated and then embedded in Araldite/Epon (Electron Microscopy Sciences, Ft. Washington, PA) and sectioned into semithin sections, which were stained with toluidine blue. Then ultrathin sections were produced. The ultrathin sections were contrasted with uranyl acetate and lead citrate and then observed on a Zeiss 10CA transmission electron microscope.

Statistical Analysis

Each treatment in the experiment was repeated at least twice. One-way ANOVA was utilized for comparisons of treatment means, such as cell number. The chi-square test was used for analysis of differences in the case of percentage comparison, such as rate of development. Significant difference was defined as P < 0.05, and no difference was defined as P > 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
H2O2-Induced Zygotic Cell Death Characterized by Morphology and TUNEL Stain

In control culture, B6C3F1 mouse zygotes cleaved normally (95%) at Day 2 and developed to blastocysts (92%) by Day 4 (Fig. 2). In developed blastocysts, an average of 2 apoptotic cells were observed by TUNEL stain (Fig. 3D), a result similar to that shown previously [43]. As it has been recognized that blastocysts exhibit PCD [32, 33, 43, 50], TUNEL-positive staining from blastocysts treated with H2O2 for 1 h were used as a positive control to confirm the effectiveness of this apoptotic assay applied in zygotes. Twenty-four hours after treatments, the total cell number (55 ± 19) was significantly (P < 0.05) reduced and percentage (25 ± 34%) of apoptotic cells significantly increased in blastocysts (n = 11) treated with 200 µM H2O2, compared to values for control, untreated blastocysts (106 ± 14 and 2 ± 2%, respectively, n = 14). However, 100 µM H2O2 treatment did not affect total as well as apoptotic cells in the blastocysts (112 ± 23 and 2 ± 3%, respectively, n = 11). In preliminary experiments, the effects of H2O2 on cleavage and development of zygotes or two-cell mouse embryos showed concentration and time dependence (data not shown).



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  		FIG. 2. Effects of H2O2 on development of mouse zygotes collected at Day 1. In untreated control culture, an average of 95% zygotes (n = 95) cleaved at Day 2, and 92% developed to blastocysts. Treatment of zygotes (n = 90) with 200 µM H2O2 inhibited cleavage and development, causing morphological hallmarks of apoptotic cell death, including cell shrinkage and membrane blebbing or cytoplasmic vacuoles



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  		FIG. 3. TUNEL stain of cell death in mouse zygotes. In the negative control, the enzyme terminal deoxynucleotidyl transferase was not added. Top panels show negative control without enzyme added (A), TUNEL-negative stain in 200 µM H2O2-treated (15 min) zygotes (B), but positive stain (green fluorescence with fluorescein isothiocyanate filter) in 1 mM H2O2-treated (1.5 h) zygotes (C) within 24 h after treatment. Lower panels: TUNEL stain at Day 4 in a developed blastocyst as in control group (D) and 200 µM H2O2-arrested one-cell (E). Arrowhead indicates positive TUNEL nuclei, and arrow indicates polar body

Mild treatment of zygotes with 200 µM H2O2 for 15 min completely inhibited cleavage, and zygotes arrested at the one-cell stage thereafter (Fig. 2). Treated zygotes exhibited shrunken morphology, but failed to display PI-positive staining 24 h after treatment, when pronuclei were not condensed. Only 31% zygotes showed PI-positive stain at 48 h after treatment. TUNEL assay did not reveal DNA fragmentation in pronuclei over 48 h (Table 1). By 72 h after treatment, pronuclei were positively stained with PI, and 46% of zygotes showed weak TUNEL staining. In contrast, intensive treatment of zygotes with 1 mM H2O2 for 1.5 h induced shrunken pronuclei and fragmented DNA detected by TUNEL stain as early as 5 h, and more evidently by 24 h after treatment (Fig. 3, C, C', C''). At 5 h, zygotes underwent degeneration characterized by shrinkage of cytoplasm, membrane permeability to PI, and condensation of pronuclei (Fig. 4). Table 1 shows that cell death occurred in zygotes as early as 5 h after intensive oxidative stress (1 mM H2O2 for 1.5 h), while obvious cell death did not occur until 48 h later in zygotes exposed to mild oxidative stress (200 µM H2O2 for 15 min). Moreover, the disruption of development and induction of cell death resulted from H2O2 itself because catalase, a decomposer of H2O2, could completely reverse this effect (data not shown).


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  		TABLE 1. H2O2-Induced cell death in mouse zygotes



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  		FIG. 4. Zygotes at 5 h after treatment with 1 mM H2O2 for 1.5 h displayed cell shrinkage (A) and membrane permeability to PI (B). PN, Pronucleus; PB, polar body

Cytochrome c Release and Caspase Activation

We further sought to determine whether cell death of zygotes induced by mild and intense oxidative stress differed in critical biochemical hallmarks, including cytochrome c release and caspase activation. Experiments were replicated three times, with at least 50 embryos observed in each treatment. H2O2 treatment of 200 µM for 15 min or 1 mM for 1.5 h induced different dynamics of cytochrome c release and caspase activation. Immunostaining showed that normal zygotes displayed reticulate and punctate staining for cytochrome c (Fig. 5B), coincident with localization of mitochondria ([45, 46], unpublished results). Zygotes treated with 1 mM H2O2 for 1.5 h almost invariably exhibited a diffuse distribution of cytochrome c staining, indicative of its release from mitochondria, from 1, 2, 4, and 6 h after treatment until the end of the observation period at 24 h (Fig. 5A). Treatment with 200 µM H2O2 for 15 min did not induce cytochrome c release until 72 h after treatment, when clumps of mitochondria and some degree of homogenous cytochrome c distribution appeared in the cytoplasm. Prior to 48 h, there were no detectable changes in cytochrome c localization, although some clumping of mitochondria was noted by JC-1 staining and by ultrastructural observation, as shown below.



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  		FIG. 5. Fluorescence micrograph showing homogenous distribution of cytochrome c staining, indicating cytochrome c release in zygotes treated with 1 mM H2O2 for 1.5 h (A), and punctuate localization of cytochrome c in mitochondria in control zygotes (B)

Similarly, 200 µM H2O2 did not induce caspase activation in zygotes within 48 h after treatment. However, 1 mM H2O2 triggered caspase activation as early as 3 h, and active caspase was detected over 24 h. In control, untreated zygotes, low caspase activity was characterized by light yellow-brownish staining, shown in a homogeneous gray appearance (Fig. 6A). The brown or dark brown stain of cytoplasm (early stage) or pronuclei (late stage) indicated active caspase after apoptotic treatment, shown in black in Figure 6, B and C. Similarly, active caspase in the cytoplasm and later in the nuclei was detected in apoptotic oocytes by fluorescence analysis [30]. Treatment with 1 mM H2O2 also has been demonstrated to induce activation of caspase 3-like protease in PC 12 cells [41]. Figure 6D shows active caspase stain in blastocysts as a positive control. Indeed, expression of caspase was detected in blastocysts [32, 51]. The active caspase seemed to be correlated with DNA fragmentation in blastocysts.



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  		FIG. 6. Caspase activity assay. A) Normal zygotes with only slight background staining, indicating absence of caspase activation. B and C) Brown staining suggestion of active caspase in 1 mM H2O2-treated zygotes; very dark brown staining in pronuclei, indicated by two arrows. D) Control staining in a blastocyst (two arrowheads indicate active caspase stain in two cells)

Changes in MMP and Distribution of Active Mitochondria

Mild oxidative stress-induced zygotic death appeared more interesting because the prolonged cell cycle arrest mimics cell death of senescent human eggs and because aging is associated with mild oxidation, rather than acute intense stress. We sought to determine whether MMP, distribution, and structure also were affected by mild H2O2 treatment, as has been reported in PCD in many somatic cell types. Changes in MMP, assessed by the shift in fluorescence emission and intensity of the dye JC-1, were compared between control untreated zygotes and zygotes treated with 200 µM H2O2 1, 2, 3, 4, and 6 h after treatment. In control, normal zygotes preloaded with JC-1, mitochondrial populations with high energy were distributed evenly throughout the cytoplasm, appearing as red fluorescence (Fig. 7, A and B). Living zygotes, like other cells, exhibit both depolarized and hyperpolarized mitochondria [13, 48]. After exposure to 200 µM H2O2 for 15 min, the MMP depolarized rapidly, as shown by the dominant green fluorescence in the intermediate region of the cytoplasm, as well as disappearance of red fluorescence and appearance of orange aggregates around cortical regions. Beginning at 2 h after treatment, the pixel ratio intensity, which reflects the MMP, was lower in the intermediate region of H2O2-treated embryos than in that of control embryos (Fig. 7C). Perinuclear distribution of active mitochondria was observed in control zygotes, but not in H2O2-treated zygotes (Fig. 8).



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  		FIG. 7. Confocal microscopy imaging of mitochondria in mouse zygotes stained with JC-1. A) Channel 1 with red (hyperpolarized, J aggregates), channel 2 with green (monomer form of JC-1), and merged fluorescence (for ratio image analysis) in control cultured zygotes and 200 µM H2O2-treated zygotes at 4 h. B) Higher magnification in the central cytoplasm of zygotes. PN, Pronucleus. C) The ratio imaging analysis collected from the confocal images. The average of relative MMP from control zygotes at each time point was set at 100%, and the MMP in treated zygotes were expressed relative to control for that time point. MMP in zygotes declined from 2 h after treatment of 200 µM H2O2 for 15 min and continued to drop over the subsequent hours. Data are presented as means ± SD in three replicates



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  		FIG. 8. MitoTracker staining of active mitochondria in zygotes. Peripronuclear distribution of mitochondria was seen (arrow) in control zygotes but absent (arrowhead) around the pronuclei (PN) in zygotes treated with 200 µM H2O2. Under differential interference contrast imaging, the pronuclei could be seen intact in both group zygotes. Different embryos were visualized under differential interference contrast and fluorescence

Ultrastructural Observation of Mitochondria

In untreated, control zygotes (Fig. 9), the sphere-shaped, vacuolated, immature mitochondria exhibited electron-dense matrices without obvious cristae. Mitochondria were scattered throughout the intermediate region of the cytoplasm (Fig. 9, upper panels). In zygotes treated with 200 µM H2O2, alterations of mitochondrial structure, including disruption of the matrix, were noted at 2 h. By 4 h after treatment, further loss of matrix and increased vacuole size were found (Fig. 9, B and D). It appeared that the membrane was damaged in some mitochondria. Furthermore, mitochondria aggregated within the cytoplasm (Fig. 9, upper panel). We did not see obvious structural changes in other organelles, including nuclear envelope and nucleoli.



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  		FIG. 9. Electron micrographs of mitochondrial ultrastructure of mouse zygotes treated with 200 µM H2O2. Top: Mitochondrial homogenous distribution in the intermediate region of control zygotes and aggregation of mitochondria in the cytoplasm in H2O2-treated zygotes. x4000. A, C) Control zygotes; B, D) H2O2-treated zygotes showing alteration of mitochondria ultrastructure in matrix or membrane. x34 000


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data demonstrate that H2O2 induces cell cycle arrest and cellular changes consistent with PCD in mouse zygotes and that mitochondria are involved in these oxidative stress-induced processes.

Oxidative Stress-Induced Zygotic Cell Cycle Arrest, Apoptosis, and/or Necrosis?

Mild oxidation treatment of zygotes initially led to decline in MMP, alteration of mitochondrial matrix, and change in the cellular distribution of mitochondria. Morphologically, the zygotes shrank but did not show the typical biochemical hallmarks of PCD, such as cytochrome c release, caspase activation, and DNA fragmentation (TUNEL-positive stain) during the first 48 h after treatment. Rather, only after 72 h of developmental arrest did they exhibit nuclear DNA fragmentation. Extensive evidence has documented that H2O2 induces apoptosis in a variety of cell systems and also acts as a second messenger during apoptotic signal transduction [7, 8, 52, 53]. The discovery that the anti-apoptotic Bcl-2 gene product has antioxidant properties further supports the importance of oxidative events in apoptosis [8, 9]. We propose that mitochondrial dysfunction may contribute to both cell cycle arrest and apoptosis during early embryo development. In fact, cell cycle arrest and apoptosis may be interconnected, and the G1->S transition of the cell cycle is the most susceptible point for cells to implement a death program [54]. Although we did not determine the specific stage of cell cycle arrest in this experiment, mitotic phase was not observed in zygotes even 72 h after H2O2 treatment.

H2O2 has been shown to induce either apoptosis or features of senescence, depending on the concentration employed or the cell lines treated. Higher concentrations of H2O2 increase the proportion of cells undergoing apoptosis [55]. Before the induction of apoptosis, many cells enter a transient phase of cell cycle arrest. Low levels of H2O2 induce many features of senescence in vitro, including cell cycle arrest, inhibition of DNA synthesis, and induction of single-strand breaks in DNA [36, 55–57]. In several mammalian cell lines, H2O2 at concentrations between 150 and 300 µM inhibit cell division, whereas high concentrations of H2O2 (>= 1 mM) induce cell death with features of both apoptosis and necrosis [58]. The present experiments with mouse zygotes demonstrated similar dose-dependent effects of H2O2 on cell death in zygotes.

Treatment of mouse zygotes with high-dose H2O2 led to early cell shrinkage, membrane blebbing, and pronuclear condensation, consistent with the classical morphological definition of apoptosis [18, 19]. Moreover, shortly after treatment, cytochrome c was released and caspase was activated, followed by DNA fragmentation in pronuclei. Intensive oxidation also might result in necrosis in zygotes as shown by their permeability to PI stain, indicative of membrane disruption. Nevertheless, swollen cells, the typical morphological change of necrosis, never were observed after intensive oxidative treatment of zygotes. Instead, shrinkage of cells appeared consistently. During necrosis, the loss of oxidative phosphorylation leads to loss of mitochondrial volume homeostasis and acute depletion of mitochondrial-derived ATP [59]. ATP is required for the orchestrated destruction of the cell that characterizes apoptosis, including cytochrome c-induced caspase activation [60]. The cytochrome c release and caspase activation observed in H2O2-treated zygotes suggests that ATP was available at least for the initial phase. Presumably as intracellular ATP is depleted, cell death shifts from an apoptotic to a necrotic form of cell death [61]. More evidence suggests that the two forms of cell death share similar signaling pathway and execution events, and that the necrosis may not be a passive response to extensive damage, as demonstrated by H2O2 treatment of some somatic cells [62–65].

Together, depending on concentration and/or exposure time, H2O2 may induce cell cycle arrest, apoptosis, or necrosis in early mammalian embryos. Intensive treatment with H2O2 induced PCD, while mild treatment induced cell cycle arrest followed by delayed PCD. Interestingly, the cell cycle arrest and apoptosis observed after mild H2O2 treatment mimics the behavior of apoptotic embryos from older women. At present, it is unclear whether the pharmacologically induced cellular events we described here underlie the decreased developmental potential of fertilized oocytes from older infertile women. It might be possible that some apoptotic machinery in the oxidative model participate in determining embryo viability.

Mitochondrial Involvement in Cell Death in Zygotes

Mitochondria play a crucial role in the early stages of apoptosis. In many systems, the release of pro-apoptotic factors, such as cytochrome c or apoptosis-inducing factor, from the mitochondrial intermembrane space into the cytosol has been demonstrated to be a primary event in caspase activation and nuclear apoptosis [14, 44, 60, 66–68]. Both the loss of outer mitochondrial membrane integrity, leading to cytochrome c release, and inner membrane depolarization are caspase-activating agents that trigger the apoptotic cascade downstream of Bcl-XL [63].

The present study showed that intensive H2O2 treatment induced cytochrome c release and activation of caspase-3, followed by nuclear DNA fragmentation, with a time course suggesting that cytochrome c release is an early event in apoptosis in the mouse zygote. Recently, caspase activity in mouse zygotes also was detected after treatment with staurosporine [51]. Cytochrome c release and caspase activation also have been observed in H2O2-induced apoptosis in somatic cells [69]. Western blot assay has been extensively used for detection of cytochrome c localization in cytosol versus mitochondria. Yet there is no reliable method available to separate mitochondria from cytosol in zygotes. By using immunocytochemistry [45, 46], we directly observed cytochrome c release from mitochondria in mouse zygotes. These data in mammalian embryos provide further evidence to confirm the importance of early cytochrome c release from mitochondria in the PCD in early embryos, even though mitochondria are relatively immature.

Disruption of the outer mitochondrial membrane could precede the loss of MMP [47]. Mitochondrial cytochrome c release could occur in the absence of mitochondrial depolarization, and collapse of the inner mitochondrial transmembrane potential might not be required for apoptosis in some types of cells [66, 67, 69, 70]. Cytochrome c may exit mitochondria via a different route, e.g., the voltage-dependent anion channel [71], than the mitochondrial permeability transition pore (MPT). Thus, in certain systems, cytochrome c release and apoptosis could be independent of the permeability transition pore [45]. Oxidative stress can change MMP and MPT ([11, 72], present study with mild oxidative stress). Whether MPT is the main pathway involved in the oxidative stress-induced cell death in the zygote needs further investigation.

On the ultrastructural level, zygote mitochondria appear as small, vacuolated, electron-dense matrix structures as observed previously [23]. These fine structures are associated with the low oxygen consumption and ATP production [24]. After oxidative stress by mild H2O2 treatment, mitochondria aggregated in the cytoplasm, and the matrix and/or membrane was disrupted. Moreover, we also observed a shift in distribution of active mitochondria after H2O2 treatment, i.e., a lack of perinuclear clustering of active mitochondria, consistent with malfunction of mitochondria, which may result in the failure in mitotic entry.

To summarize, both mild and intensive oxidative stress induced cell death of zygotes. Mild oxidative stress induced cell cycle arrest, followed by delayed apoptotic cell death, possibly through altering mitochondrial activity and structures; intensive oxidative stress probably triggered apoptosis and terminal necrosis through early cytochrome c release from mitochondria and caspase activation. Therefore, the present study provides evidence that mitochondria are involved in the oxidative stress-induced cell cycle arrest and cell death in a single cell system, mammalian zygotes.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. Peter Smith for his valuable discussion and encouragement and Mr. Louis Kerr for his enthusiastic assistance with electronic microscopy.


   FOOTNOTES
 
First decision: 24 November 1999.

1 This work was supported in part by the National Institute of Health (NIH K081099) and Women and Infants Hospital/Brown Faculty Research Fund. Back

2 Correspondence: David Keefe, Dept. of Ob-Gyn, Women & Infants Hospital, Brown University, 101 Dudley Street, Providence, RI 02905. FAX: 401 453 7599; dkeefe{at}smtp.wihri.org Back

Accepted: January 27, 2000.

Received: October 27, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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