BIOLOGY OF REPRODUCTION 62, 1828–1834 (2000)
Cytoplasm Mediates Both Development and Oxidation-Induced Apoptotic Cell
Death in Mouse Zygotes1
Lin Liu4 and David L. Keefe2,3
Department of Obstetrics/Gynecology, Brown University, Women and Infants Hospital, Providence,
Rhode Island 02905
Marine Biological Laboratory, Woods Hole, Massachusetts 02543
ABSTRACT
Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged
women abrogates the effects of age on fertility. Oxidative stress,
mitochondrial dysfunction, and apoptosis are associated with
senescence. To develop an animal model of egg senescence, we
treated mouse zygotes with 175 mM H2O2 that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed
zygotes with nuclei and cytoplasm from treated or untreated
zygotes, then followed development and apoptotic cell death in
the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls.
Rates of cleavage and development to morula and blastocysts
were significantly lower (P , 0.01) in zygotes reconstituted
from untreated pronuclei and H2O2-stressed cytoplasts than
those of nuclear transfer controls. Instead, the arrested, reconstituted zygotes displayed TUNEL staining at a similar rate to
that of H2O2-treated controls, suggesting that apoptotic potential could be transferred cytoplasmically. On the other hand,
rates of cleavage and development to morula and blastocyst of
the reconstituted zygotes, derived from stressed pronuclei and
untreated cytoplasm, were significantly increased (P , 0.05),
compared to those of H2O2-treated, control zygotes, indicating
that healthy cytoplasm could partly rescue pronuclei from oxidative stress. Although oxidation stressed both nuclei and cytoplasm, cytoplasm was more sensitive than nuclei to oxidative
stress. It is suggested that cytoplasm, most likely mitochondria,
plays a central role in mediating both development and apoptotic cell death induced by oxidative stress in mouse zygotes.
INTRODUCTION
Aging-related decline in female fertility is a common
phenomenon in older women and in females from many
other long-lived mammalian species [1]. Maternal age has
been demonstrated to affect oocyte quality and early embryo development [2, 3]. Oxidative stress [4], and apoptosis
or programmed cell death (PCD) underlie oocyte and embryo dysfunction, as evidenced by the finding that nuclear
DNA fragmentation and apoptosis occur in aged mouse and
human oocytes and that apoptotic human embryos exhibit
high levels of hydrogen peroxide (H2O2) [5–7]. Oxidative
stress and mitochondrial decay are likely to be major conThis work was supported in part by the NIH (K081099) and Women and
Infants Hospital/Brown Faculty Research Fund.
2
Correspondence: David Keefe, Women & Infants Hospital, Brown University, Dept. of Ob/Gyn, 101 Dudley Street, Providence, RI 02905. FAX:
401 453 7599; e-mail: [email protected]
1
Received: 15 November 1999.
First decision: 8 December 1999.
Accepted: 26 January 2000.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
tributors to aging and aging-associated cell death [8–15].
H2O2, a form of reactive oxygen species (ROS), mediates
mitochondrial oxidative damage that may lead to mitochondrial dysfunction and ultimately cell death [16–18].
Alterations in mitochondrial structure and function were
shown to occur early during PCD or apoptosis and mitochondria appeared to be the central regulator of apoptosis
[19, 20], suggesting that oxidative stress, mitochondria, and
apoptosis may play important roles in aging.
In mammalian oocytes and early embryos, PCD occurs
by default in embryos that fail to execute essential developmental events, and genes that regulate apoptosis are expressed [21–23]. However, the contribution of nucleus versus cytoplasm to cell death in early embryos remains unclear. In the present study, we created an apoptotic cell
death model in mouse zygotes by exposing them to H2O2,
based on previous data [24]. It is assumed that nuclear
transfer (NT) might be a potential tool for dissecting the
contribution of nucleus versus cytoplasm to apoptosis and
also probably for isolating the effects of H2O2 on cytoplasm
versus the nucleus. To examine the role of cytoplasm in
mouse zygotes in mediating apoptosis, experiments were
designed to determine whether apoptotic potential could be
transmitted via cytoplasm to untreated nuclei using NT.
Further experiments were undertaken to determine whether
healthy cytoplasm can rescue nuclei at the early stage of
apoptosis. Because mitochondria in the cytoplasm are especially sensitive to the effects of oxidative stress, next we
tested the hypothesis that cytoplasm is more sensitive than
nuclei to oxidative stress by comparing the developmental
potential and apoptosis of embryos reconstituted from either treated nuclei or cytoplasm and their untreated counterparts.
MATERIALS AND METHODS
Reagents, Animals, Embryo Collection, and Culture
All reagents were purchased from Sigma Chemical Co.
(St. Louis, MO), unless stated otherwise. Pregnant mare’s
serum gonadotropin (PMSG) used for superovulation was
purchased from Calbiochem (La Jolla, CA). Animals were
cared for according to procedures approved by the Marine
Biological Laboratory and Women and Infants Hospital Animal Care Committees. Two- to three-month-old B6C3F1
female mice (Charles River Laboratory, Boston, MA) were
superovulated by intraperitoneal injection of 7.5 IU eCG,
followed 46–48 h later by injection of 7.5 IU hCG, then
mated individually with B6C3F1 males of proven fertility.
Successfully mated females 20–21 h after hCG injection
were used for collecting zygotes (Day 1). Zygotes enclosed
in cumulus masses were released from the ampullae into the
Hepes-buffered potassium simplex optimized medium
(HKSOM), with 0.03% hyaluronidase, then cumulus cells
were removed by gentle pipetting [24]. In vitro manipulation
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CYTOPLASM CONTROL OF ZYGOTIC DEATH
FIG. 1. Nuclear transfer in mouse zygotes treated with H2O2. A) A normal B6C3F1 mouse zygote. Pronuclei (PN) are seen clearly with Nomarski optics. Polar body (PB) is also indicated. B) An enucleated zygote
(cytoplast). The pipette contains the pronuclear karyoplast. C) A group of
pronuclear karyoplasts separated from normal control zygotes. D) A group
of pronuclear karyoplasts separated from H2O2-treated zygotes. Note
there is no obvious difference in morphology between C and D. E) An
enucleated cytoplast coupled with an inserted PN (couplet). F) A reconstituted zygote shortly after fusion of the pronuclear karyoplast to the
enucleated cytoplast. Note the peripheral location of the PN.
of embryos was carried out in HKSOM at 34–378C on a
heating stage. The modified KSOM, supplemented with nonessential amino acids and 2.5 mM Hepes, was used for in
vitro culture [25–27]. Embryos were washed and cultured in
50-ml droplets of KSOM under mineral oil at 378C in a
humidified atmosphere of 7% CO2 in air. In this culture system, blastocysts, derived from in vitro culture for 3 days of
fertilized eggs, developed to term (70%, 33/47) equally as
well as in vivo blastocysts (75%) after embryo transfer into
pseudopregnant Day 3 CD-1 foster females. Embryos were
assessed for cleavage at Days 2 and 3, and for development
to blastocyst at Day 4. The number of apoptotic cells was
determined by TUNEL (TdT-mediated dUTP nick end labeling) assay in some embryos at Day 4.
Detection of Apoptosis by TUNEL Assay
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 propidium iodide (PI, 50 mg/ml;
Molecular Probes, Eugene, OR), as described previously
[28, 29]. Fluorescence was detected using either a Zeiss
LSM 510 laser scanning confocal microscope (Germany)
or Zeiss Axiovert 100TV inverted fluorescence microscope.
In addition, plasma membrane blebs, cell shrinkage, and
nuclear condensation also were measured as indicative of
apoptosis [22].
Detection of Mitochondrial Membrane Potential
Mitochondrial membrane potential was measured by the
lipophilic cationic probe 5,59,6,69-tetrachloro-1,193,39-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) as described in previous reports [30]. Differences in mitochondrial membrane potential shift fluorescence emission and intensity of the dye JC-1. In the presence of a high mitochondrial membrane potential, JC-1
forms J-aggregates that emit red fluorescence, while JC-1
in its monomeric form emits green fluorescence at low mitochondrial membrane potential. Both colors can be de-
1829
FIG. 2. Development in vitro of untreated, culture control zygotes and
NT control zygotes, but developmental arrest and cell death of zygotes
exposed to 175 mM H2O2 for 13 min.
tected using the confocal microscope (Zeiss LSM510, Germany) 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 ml HKSOM containing
1.25 mM JC-1 for 20 min at 378C. The ratio analysis was
performed with Zeiss LSM510 software and MetaMorph
imaging software (Universal Imaging Corporation, Cambridge, MA).
Transmission Electron Microscopy
Embryos were fixed in 2.5% glutaraldehyde in 0.1 M
phosphate buffer at 48C 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, dehydrated, and then embedded in Araldite/
Epon, and sectioned into semisections (1 mm), that were
stained with toluidine blue. Ultrathin sections were cut with
a diamond knife. Ultrathin sections (0.1 mm) were contrasted with uranyl acetate and lead citrate and observed on a
Zeiss 10CA transmission electron microscope (Germany).
Nuclear Transfer
Pronuclei exchanges between zygotes and the reconstitution of zygotes were achieved by NT and electrofusion
[31, 32]. Zygotes in the experimental and NT controls were
incubated for 20 min in the HKSOM, supplemented with
cytochalasin B (5 mg/ml) and nocodazole (2 mg/ml) to render the membrane sufficiently elastic to withstand possible
damage by an enucleation micropipette. Enucleation and
insertion of pronuclei (karyoplast) (Fig. 1, A–E) were performed on an inverted microscope (Zeiss Axiovert 100TV,
Germany) with Nomarski optics and Narishige micromanipulators (Japan). Pronuclear karyoplasts, coupled with
enucleated cytoplasts (couplets), were induced to fuse by
applying an AC pulse (5 V) for 2–3 sec, followed by a
single DC pulse of 1.2 kV/cm for 60 msec, with a BTX
Electro Cell Manipulator (ECM 2001, San Diego, CA). One
hour later, fusion was checked and the fused (reconstituted)
zygotes (Fig. 1F) were continuously cultured in KSOM.
Nuclear Transfer Experimental Design
Preliminary dose–response experiments showed that 175
mM H2O2 treatment for 13 min in culture was the minimal
dose-duration that reliably induced cell cycle arrest and apoptosis in zygotes, so this dose-duration was used in the
following experiments. Control zygotes were cultured with-
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LIU AND KEEFE
out exposure to treatments. Another control group underwent NT, but no pharmacotherapy. All treatments were included in each of the following experiments.
I. To test the hypothesis that cytoplasmic factors mediate
apoptosis, normal untreated karyoplasts or pronuclei
(PN) were transferred to cytoplasts derived from H2O2treated zygotes. The PN exchange between untreated
zygotes served as NT controls. Based on the timing of
functional and structural changes in mitochondria, the
timing of NT and fusion-induced reconstitution after
H2O2 treatment was chosen at 1–1.5 h and 3.5–4 h,
respectively (Fig. 4I).
II. To test the hypothesis that pronuclei from zygotes that
had been exposed to H2O2 could be rescued to develop
further by fusion with healthy cytoplasm, karyoplasts
from H2O2-treated zygotes were transferred to normal,
untreated, enucleated cytoplasts. Again, based on the
timing of functional and structural changes in mitochondria, the timing of NT and fusion-induced reconstitution after H2O2 treatment was chosen at 1–1.5 h
and 3.5–4 h, respectively (Fig. 5II).
III. Because cytoplasm, especially mitochondria are sensitive to ROS, and cytoplasm regulates PCD in many cell
types, we hypothesized that cytoplasts would be more
sensitive than karyoplasts to oxidative stress. To test
this hypothesis, the relative sensitivity to H2O2 of nuclei versus cytoplasm from zygotes was compared directly by creating pronuclear karyoplasts and cytoplasts
by micromanipulation, treating each separately with
175 mM H2O2 for 13 min, or vehicle (KSOM), reconstituting treated with untreated counterpart, then observing cleavage and developmental rates, morphology,
and TUNEL staining of reconstituted zygotes (Fig.
6III).
Statistical Analysis
Each experiment was repeated at least four times. Pooled
data derived from rates of development or cell death were
expressed as the percentage of total zygotes treated. Percentages were transformed using arcsin transformation.
Transformed data were analyzed by ANOVA and means
compared by Fisher’s protected least-significant difference
(PLSD) using the StatView software (1998) from SAS Institute Inc. (Cary, NC). Comparisons of treatment means,
e.g., cell number, were carried out by ANOVA. When a
significant effect was detected, differences between means
were analyzed by Fisher’s PLSD. Significant differences
were defined as P , 0.05.
RESULTS
During in vitro culture, 97% of cultured control zygotes
(n 5 298) cleaved at Day 2 and 96% developed to blastocyst (90%) and morulae (6%) by Day 4, whereas only
9% of zygotes (n 5 290) exposed to H2O2 cleaved and 6%
reached morulae and blastocyst stages of development by
Day 4. Indeed, nearly 90% of H2O2-treated zygotes arrested
at the one-cell stage, demonstrating that H2O2 induced cell
cycle arrest and inhibited development of zygotes (Fig. 2).
Nuclear transfer control zygotes (n 5 173) displayed similar rates of cleavage at Day 2 (99%) and morula and blastocyst development (93%) at Day 4 to those of embryo
culture controls. The data presented are pooled from several
experiments. Blastocysts developed from NT control zygotes appeared morphologically similar to those of culture
FIG. 3. Alterations in mitochondrial function and structure in mouse
zygotes treated with 175 mM H2O2. A) The average relative ratios (red/
green channel) (mean 6 SD) derived from JC-1 stain and confocal imaging, indicative of mitochondrial membrane potential, in control zygotes
was considered 100%. **Indicates significant difference (P , 0.01) between control and H2O2 groups. B) Electron micrographs of mitochondrial
ultrastructure. In the control zygotes, electron-dense matrix mitochondria
are widely distributed, whereas disruption of matrix and membrane of
aggregated mitochondria was observed in H2O2-treated zygotes. Arrow
indicates mitochondrial matrix. 315 750.
control zygotes that had not been subjected to micromanipulation, except that early hatching was observed in some
blastocysts derived from NT zygotes (Fig. 2). Furthermore,
high fusion rates (.90%) were obtained consistently
throughout the experiments, regardless of oxidative stress
on either pronuclear karyoplasts or cytoplasts. Obviously,
H2O2 did not affect plasma membrane integrity in terms of
membrane fusion.
H2O2-Induced Alterations in Mitochondrial
Function and Structure
Cell shrinkage and developmental arrest were observed
in zygotes exposed to H2O2. Further experiments showed
that mitochondrial function, assessed by mitochondrial
membrane potential with JC-1 and confocal microscopy,
was impaired in zygotes treated with H2O2. The relative
ratio pixel intensity, indicative of mitochondrial membrane
potential, was significantly (P , 0.01) lower in zygotes 3
h and 6 h after H2O2 treatment, compared to controls (Fig.
3A).
CYTOPLASM CONTROL OF ZYGOTIC DEATH
1831
At the ultrastructural level, control zygotes exhibited
sphere-shaped, vacuolated, immature mitochondria, with
electron-dense matrices that lacked obvious cristae (Fig.
3B). Mitochondria were scattered in the intermediate region
of the cytoplasm. Treatment with 175 mM H2O2 altered
mitochondrial structures 3 h and 4 h later. Typically, the
electron-dense matrix appeared disrupted, and membranes
of some mitochondria were damaged, leading to increased
vacuole size and mitochondrial swelling. Moreover, mitochondrial aggregation was found. We did not see obvious
structural changes in other organelles, including the nuclear
envelope or nucleolus.
Oxidative-Stressed Cytoplasm Transmits
Apoptotic Potential
In Experiment I, NT control zygotes (n 5 85) cleaved
(99%) by Day 2 and developed to morula (18%) and blastocysts (75%) by Day 4 (Fig. 4A). In contrast, zygotes reconstituted 3.5–4 h after H2O2 treatment from untreated
pronuclei and cytoplasts derived from H2O2-treated zygotes
(H2O2 cyto, n 5 82) exhibited significantly lower (P ,
0.01) rates of cleavage (10%) and development (9%) to
morula and blastocysts, but similar to those of H2O2-treated
controls. It is thus suggested that the effects of oxidative
stress could be transferred cytoplasmically.
Next, we separated nuclei from cytoplasm immediately
after exposure to H2O2 and reconstituted them 1–1.5 h after
treatment with normal pronuclei from untreated zygotes to
minimize oxidative effects on the cytoplasm. Although NT
control zygotes (n 5 88) in this experiment exhibited similar high rates of cleavage (100%) and development to morula and blastocyst (93%), reconstituted zygotes (n 5 87)
derived from untreated karyoplasts fused with cytoplasm
that had been exposed to H2O2, exhibited significantly delayed development in culture. Only 48% of these reconstituted zygotes cleaved and 52% of these reached morula
(40%) and blastocyst stages by Day 4 (Fig. 4B). It appeared
that the early separation of oxidatively stressed cytoplasm
and fusion with untreated pronuclei significantly improved
cleavage of reconstituted zygotes (compare Fig. 4A with
Fig. 4B). However, the H2O2 cyto group exhibited a similar
rate of development to blastocyst to that of H2O2-treated
control group (12% versus 5%, P . 0.05). Furthermore,
the rate of nuclear DNA fragmentation in the arrested zygotes did not differ between the H2O2 cyto and the H2O2treated control groups (14% versus 25%, P . 0.05; Fig.
4C). Taken together, these experiments indicated that H2O2stressed cytoplasm induced morphology and TUNEL staining in reconstituted zygotes consistent with apoptosis, even
though the nucleus itself never had been exposed to H2O2.
That cytoplasm from zygotes treated with H2O2 can transfer
the apoptosis program to zygotes reconstituted from untreated nuclei supports an important role for cytoplasm in
the regulation of apoptosis in embryos.
Normal Cytoplasm Can Rescue Some Pronuclei
from Oxidative-Stressed Zygotes
In Experiment II, compared to those of H2O2-treated
control zyogtes (n 5 164), rates of cleavage (6%) at Day
2 and development to blastocyst (4%) at Day 4 were not
improved in the reconstituted, H2O2 PN zygotes (n 5 100),
derived from H2O2-stressed pronuclei, fused at 3.5–4 h with
untreated cytoplasm. However, rates of cleavage to two to
four cells (33%) at Day 3 and development to morula
(18%) at Day 4 were significantly increased (Fig. 5A).
FIG. 4. Cytoplasm transmits apoptotic potential (Experiment I). A) Pronuclei were removed from zygotic cytoplasm at 2 h and fusion of the
couplets started 3.5–4 h after H 2O2 treatment of zygotes. B) Pronuclei
were removed from zygotic cytoplasm right after treatment and fusion of
the couplets started 1–1.5 h after H 2O2 treatment of zygotes. Nuclear
transfer control: pronuclei (PN) separated from untreated, normal zygotes
were reconstituted with untreated cytoplasm (cyto); H2O2 cyto: untreated
PN were reconstituted with cytoplasm separated from zygotes treated
with 175 mM H2O2. *Indicates significant difference (P , 0.05) between
NT control and H2O2 cyto groups. C) DNA fragmentation in arrested zygotes was determined by TUNEL assay.
These results indicated that H2O2 also stressed pronuclei
and that delayed cleavage could occur in zygotes with oxidative-stressed pronuclei. To shorten the duration of possible oxidative stress effects on pronuclei, pronuclei were
removed from oxidative-stressed zygotes immediately after
exposure to H2O2 and reconstituted 1–1.5 h after treatment
with normal cytoplasm derived from untreated zygotes. As
a result, rates of cleavage (33%) at Day 2 and development
to morula (15%) and blastocyst (21%) at Day 4 of the reconstituted zygotes with H2O2 stressed pronuclei (n 5 114)
were significantly increased (P , 0.05), compared to those
of H2O2-treated control zygotes (n 5 126) (Fig. 5B).
Furthermore, the total cell number and the number of
apoptotic cells did not differ statistically (P . 0.05) in blastocysts derived from NT control, reconstituted zygotes with
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LIU AND KEEFE
FIG. 6. Cytoplasm is more susceptible to oxidative stress than pronuclei
(Experiment III). Pronuclear karyoplasts and cytoplasts were separately
treated with 175 mM H2O2 for 13 min and fused with their counterparts
2.5 h after treatment. H2O2 control: zygotes were treated with H2O2 in
KSOM for 13 min; NT control: pronuclei (PN) separated from untreated
normal zygotes were reconstituted with untreated cytoplasm (cyto); H2O2
PN: H2O2-treated PN were transferred into untreated, normal cytoplasts;
H2O2 cyto: H2O2-treated cytoplasts were fused with untreated, normal PN.
Cytoplasm Is More Sensitive Than Nuclei
to Oxidative Stress
FIG. 5. Rescue of pronuclei from oxidative-stressed zygotes by transfer
into untreated, normal cytoplasm (Experiment II). A) Pronuclei were removed from zygotic cytoplasm at 2 h and fusion of the couplets started
3.5–4 h after H 2O2 treatment of zygotes. B) Pronuclei were removed from
zygotic cytoplasm right after treatment and fusion of the couplets started
1.5–2 h after H 2O2 treatment of zygotes. H2O2 control: zygotes were treated with 175 mM H2O2 for 13 min, then cultured in vitro; H2O2 PN: Pronuclei were separated from H2O2-treated zygotes and transferred into untreated cytoplasm, separated from normal zygotes. *Indicates significant
difference (P , 0.05) between H2O2 control and H2O2 PN groups. C)
Comparisons of the total cell number as well as the number of apoptotic
cells in blastocysts derived from in vitro culture of normal zygotes, NT
control zygotes, or zygotes reconstituted 1–1.5 h from oxidative-stressed
pronuclei and normal cytoplasm (H2O2 PN). The observed number of
blastocysts is shown in the bars. No significant differences in the cell
number were observed among the groups (P . 0.05).
H2O2-stressed pronuclei, and culture control zygotes (Fig.
5C). Moreover, fewer arrested, reconstituted zygotes with
H2O2-stressed pronuclei exhibited DNA fragmentation than
H2O2 treatment control (9% versus 25%, P , 0.01). The
results demonstrated that some oxidative-stressed pronuclei
could be partially rescued by early separation from oxidative-stressed cytoplasm and fusion with untreated, healthy
cytoplasm. Comparison of the results from the above two
experiments (Fig. 5, A and B) suggests that nuclear–cytoplasm interaction may amplify the cell arrest and death induced by oxidative stress.
In Experiment III, the relative sensitivities of the nucleus
and cytoplasm were compared directly. As in other experiments, H2O2 almost completely inhibited cleavage of zygotes
(n 5 50). No zygotes (n 5 45) cleaved if normal, untreated
pronuclei had been reconstituted with H2O2-treated cytoplasts
(Fig. 6). In contrast, all zygotes (n 5 49) reconstituted from
untreated cytoplasts and untreated pronuclei cleaved on Day
2 and 92% of these developed to morula and blastocyst by
Day 4. Twenty and 77% of zygotes (n 5 45) reconstituted
from untreated cytoplasts and treated pronuclei cleaved by
Day 2 and Day 3, respectively, and 47% of zygotes developed
to morula and blastocyst stages by Day 4. Blastocyst development was however very low at only around 10%. The TUNEL assay showed that 13% (n 5 30) of arrested zygotes,
reconstituted from H2O2-treated cytoplasts, exhibited evidence
of apoptotic cell death, similar to the rate (18%, n 5 33) found
in H2O2 treatment control zygotes (P . 0.05). In addition,
cell shrinkage, membrane blebbing, and cytoplasm vacuolization, morphologic characteristics of apoptosis [22], were observed in H2O2-treated zygotes or cytoplasm. Therefore, not
only could treated cytoplasm transfer the apoptosis program,
and untreated cytoplasm partially rescue treated pronuclei, but
also cytoplasm was more sensitive than nuclei to the apoptosis-inducing effects of H2O2.
DISCUSSION
Extensive evidence has been reported that H2O2 either directly induces or acts as a second messenger to induce apoptosis in a variety of cell systems [13, 14, 16, 33, 34]. Present
experiments demonstrate that oxidation-induced cell death by
H2O2 also occurs in mammalian eggs, further supporting the
CYTOPLASM CONTROL OF ZYGOTIC DEATH
general rule of oxidative stress-induced cell death. The disruption of development and induction of cell death must have
resulted from H2O2 itself because catalase, a decomposer of
H2O2, could completely reverse this effect (data not shown).
Oxidative-stressed zygotes initially were arrested developmentally, then underwent apoptotic cell death, as evidenced
by cell shrinkage and DNA fragmentation. Low levels of
H2O2 are important products of normal metabolism and are
not allowed to accumulate to harmful concentrations normally. However, when the mitochondrial antioxidant defense
mechanisms are compromised, such as occurs during aging
[18], low levels of H2O2 also can induce cell cycle arrest and
senescence in somatic cells [35–38]. Indeed, cell cycle arrest
and apoptosis may be interconnected [39, 40].
Mitochondria, specifically mtDNA, have been shown to
be important targets of age-associated free radical attack
[10]. Mitochondrial DNA mutations or deletions are closely
associated with the aging process in many long-lived, postmitotic cells [41, 42]. Human oocytes harbor mtDNA deletions, and the frequency of the mtDNA deletions in oocytes increases as women age [43], implying mitochondrial
dysfunction in reproductive aging. With increasing age, mitochondria tend to be larger, with increased matrix vacuolization and shorter cristae and decreased number of dense
granules [44, 45]. Mild oxidation led to mitochondrial dysfunction, alteration of matrix and mitochondrial membranes, and appearance of swollen vacuoles in mitochondria. Voltage-dependent anion channels might have been
involved in mitochondrial matrix swelling and membrane
rupture during apoptosis [46]. Our observations of damaged
mitochondria in mouse zygotes after oxidative stress resembled changes reported in other tissues observed during aging. While acute toxicity model systems might not be relevant to apoptosis as it occurs during aging [47], the cell
cycle arrest and apoptosis induced by mild oxidation might
be an alternate model for aging-related studies [36].
Our findings in both Experiments I and III that oxidative-stressed cytoplasm could transmit apoptotic potential
to untreated healthy nuclei after reconstitution provide further evidence for an important role of cytoplasm in the
regulation of apoptosis. Cytoplasmic control over nuclear
behavior during early development has been demonstrated
previously [48]. Study of the role of cytoplasm in the regulation of development and death of reconstituted zygotes
provides a model to investigate the role of this cellular
compartment that includes mitochondria in apoptosis during early development. The regulation of apoptosis by cytoplasm reported here probably is mediated by mitochondria. Mitochondria control apoptosis in a variety of cell
types [20, 49]. As mitochondria were highly sensitive to
oxidative stress, most likely they were the main cytoplasmic components involved in the cytoplasmic transmission
of apoptotic potential. Similarly, mitochondrial defects also
can be transmitted [50, 51].
If cytoplasmic dysfunction promotes the cell death of
oxidative-stressed zygotes, then replacement of defective
cytoplasm with normal cytoplasm might benefit subsequent
embryo development. Indeed, Experiment II indicated that
some oxidative-stressed pronuclei could be rescued by reconstitution with normal healthy cytoplasm. Experiment III
also demonstrated that nuclei are less sensitive than cytoplasm to oxidative stress. Prolonging exposure of pronuclei
to oxidative-stressed cytoplasm probably amplifies the cell
death effects caused by oxidation. In other words, early
separation of oxidative-stressed pronuclei and reconstitution with healthy cytoplasm better recovered the pronuclei
1833
than later separation of them and reconstitution with
healthy cytoplasm. In fact, when zygotes were reconstituted
(Experiments IB and IIB), prior to changes in mitochondrial
function and structure, cleavage was improved significantly
in both types of reconstituted zygotes. However, development to blastocyst was improved only in zygotes reconstituted from oxidative-stressed pronuclei and untreated cytoplasm, but not from untreated pronuclei with H2O2-treated cytoplasm. This finding further suggests cytoplasmic
mediation of development and apoptosis of embryos.
That the cytoplasm was more sensitive to the effects of
H2O2 than pronuclei also was evidenced by morphology in
that the separated cytoplasm was damaged as extensively as
it had been in treated, intact zygotes. When reconstituted with
untreated cytoplasm, oxidative-stressed pronuclei, in contrast
to H2O2 control zygotes, displayed significantly improved
rates of cleavage and development to morula and blastocysts.
While oxidation can affect both nuclear and mitochondrial
DNA during aging, mtDNA seems more susceptible to oxidation damage [52–55], because it lacks the protective histones and extensive DNA repair capabilities that protect nuclear DNA [56, 57]. It may imply that cytoplasm is affected
by oxidation more intensively or earlier than nuclei.
In conclusion, cytoplasm, probably through mitochondria,
may play a central role in mediating either development, or
cell cycle arrest and apoptotic cell death induced by oxidative
stress in mouse zygotes. Based on the present NT experiments
and other research in somatic cell systems referred to above,
we propose the following model of oxidation in zygotes. During phase I, oxidation attacks cytoplasm, such as mitochondria. During phase II, cytoplasmically mediated nuclear damage occurs. Finally at phase III, both nuclei and cytoplasm
are damaged irreversibly. As oxidation and disruption of mitochondrial function are associated closely with aging [58],
exploring oxidation in development and apoptosis by using in
vitro oocytes or zygotes might be a good model to study aging
associated process, including oocyte dysfunction. Cytoplasmic
dysfunction has been hypothesized as a cause of oocyte infertility in women. Whether NT might be applied to treat such
causes of oocyte dysfunction needs further investigation. On
the other hand, in cases when nuclear damage is the major
problem, NT could carry significant risks, because most cell
types depend on the cytoplasm to execute cell death. Transfer
of defective nuclei into normal cytoplasm could interfere with
important adaptive function of apoptosis.
ACKNOWLEDGMENTS
We thank Drs. Peter Smith for providing some critical instruments,
James Trimarchi for valuable discussion, and Mr. Louis Kerr for his assistance with electronic microscopy.
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