Research ArticleExperimental Studies

Doxorubicin Induces Early Embryo Apoptosis by Inhibiting Poly(ADP ribose) Polymerase

QING-LING WANG, SHAO-CHEN SUN, JEMESBOND HAN, YONG-CHUL KWAK, NAM-HYUNG KIM and XIANG-SHUN CUI
In Vivo September 2012, 26 (5) 827-834;

Abstract

Background: The effect of Doxorubicin, a widely used chemotherapeutic agent, on early mouse embryonic development has not been previously characterized. Materials and Methods: Expression of apoptosis-related genes and poly(ADP-ribose) polymerase (PARP) family genes were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Apoptosis in mouse blastocysts was tested using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Cleaved (c)-PARP was analyzed by western blot. Results: A 20 h exposure to doxorubicin caused rapid cytoplasmic fragmentation, DNA condensation and disruption of the cytoskeleton in mouse embryos. Doxorubicin altered the expression of genes involved in DNA repair and apoptosis and blocked early embryonic development, suggesting that doxorubicin affects DNA synthesis and repair. Furthermore, the effect of doxorubicin on early embryo development was determined by assessing the rates of development to different stages and an apoptosis index. Both assays confirmed that doxorubicin altered embryonic development. In conclusion, doxorubicin blocked pre-implantation development in early mouse embryos by altering apoptosis-related gene expression and inactivating DNA repair by PARP.

Doxorubicin (an anthracycline-derived chemotherapeutic agent) is used in the treatment of cancer and is unquestionably beneficial as a therapeutic agent. The ovarian and oocyte toxicity of doxorubicin is well-documented. Consequently, the detrimental reproductive and developmental problems faced by women and their offspring cannot be ignored. Doxorubicin reduces the finite pool of primordial oocytes within the ovaries, and the damage has been correlated with the maturity of the oocytes (1, 2). Differences in the sensitivity of germinal vesicle (GV) and metaphase II (MII) oocytes to doxorubicin are reflected in the differing levels of apoptosis in oocytes of each stage (3). The characteristic cellular fragmentation in oocytes and embryos is consistent with the activation of programmed cell death, as has been proposed by several studies, based on the presence of many hallmarks of apoptosis, including DNA cleavage, activation of caspases, translocation of phosphatidylserine to the outer leaflet of the plasma membrane, and cytoplasmic condensation (4). Doxorubicin induces both cellular and DNA fragmentation of murine oocytes (5, 6), and thus represents an ideal model for studying the mechanisms of cell death. However, little is known regarding the effects of doxorubicin on early embryo development in vitro.

Doxorubicin elicits apoptosis by several mechanisms in a variety of cell types. It binds to DNA, causes the generation of free radicals that leads to DNA damage and lipid peroxidation, and inhibits enzymes such as helicases and toposoimerase II, which are important for DNA replication and transcription (7). All these actions affect DNA function in cells exposed to doxorubicin by disrupting transcription and/or replication, which eventually causes apoptosis of the cells. Apoptosis is a common feature of mammalian development and plays a fundamental role in cell growth, development and homeostasis. B-cell lymphoma/leukemia 2-like 1 (Bcl2l1) gene product acts as an essential regulator of apoptosis and may either suppress (Bcl-2, Bcl-xl) or promote (Bak, Bad) the induction of apoptosis (8-10). Caspases are cysteine proteases that are involved in programmed cell death and have recently been found to have vital functions in mouse pre-implantation embryonic development (11).

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that has a well-characterized role in base excision repair, which is one of the primary repair mechanisms to resolve DNA lesions caused by endogenous processes aswell as those caused by exogenous chemical exposure and irradiation (12). PARP also has a well-documented role in testicular germ cells (13, 14), including a role in DNA damage (15). PARP proteins are involved in the detection of strand breaks and signaling in both the base excision repair and nucleotide repair pathways (16, 17). PARP catalyzes poly(ADP-ribosylation) (PAR). The PARP family consists of 18 homologs (PARP 1-18) that have a conserved catalytic domain composed of 50-amino acid residues forming the ‘PARP signature’ (18). The best characterized member of the PARP family is PARP1, a 113-kDa enzyme encoded by the ADP-ribosyl transferase gene in humans, located on chromosome 1 (19). PARP1 contains three functional domains: a DNA-binding domain (DBD), an automodification domain (AMD), and a catalytic domain (CD). The DNA binding domain contains both zinc-fingers that bind to breaks in DNA and a nuclear localization signal (NLS) that ensures the translocation of PARP1 into the nucleus. The NLS also contains a caspase 3 cleavage site. Cleavage of PARP by caspase-3 inactivates the DNA repair ability of PARP. Cleaved PARP (c-PARP) is a marker of apoptosis. The most important role of PARP is in DNA repair, especially resolving single-strand breaks. Both PARP1 and PARP2 have been shown to function in base excision repair (20).

The aim of our study was to characterize the expression of apoptosis-related genes after doxorubicin treatment in early mouse embryos, since most previous studies have been conducted on oocytes (2-6). We focused on early pre-implantation embryos since they represent the physiological stage at which embryos might unknowingly be exposed to chemotherapy.

Materials and Methods

All chemicals were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA) unless indicated otherwise. A minimum of three independent experiments were performed for each treatment. Within each experiment, oocytes were obtained from the same group of ovaries collected on the same day.

Generation of mouse embryos and in vitro culture. Animals were treated in accordance with the institutional animal care and use guidelines and all experiments were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1985). To obtain embryos, 6-week-old ICR female mice (Samtako, Osan, Korea) were induced to superovulate by intraperitoneal injections of 5 IU pregnant mare serum gonadotropin (PMSG) (Sigma), followed by 5 IU human chorionic gonadotrophin (hCG) (Sigma), 48 h later. Each female was then caged individually with one ICR male mouse. The presence of a vaginal plug the following morning confirmed successful mating (day 1 of pregnancy). One cell (1C) zygotes were collected from the ampullae of superovulated mated females 20 h after hCG injection. Cumulus cells were removed by pipetting with 0.1 mg/ml hyarulonidase (Sigma) in M2 (Sigma) medium. The embryos were cultured in M16 culture medium in the presence or absence of 5 nM, 50 nM or 200 nM doxorubicin at 37°C in an atmosphere containing 5% CO2.

View this table:
Table I.

List of primers used for real-time RT-PCR.

Sample collection. Two-cell (2C), four-cell (4C), morula (MO) and blastocyst (BL) stage embryos were collected and then snap-frozen in liquid nitrogen and stored at −80°C until RNA was isolated for analysis by real time reverse transcription polymerase chain reaction (RT-PCR). To examine cytoskeletal changes, embryos were collected after a 20-h treatment with 50 nM doxorubicin and fixed in 3.7% paraformaldehyde in phosphate buffered saline (PBS) for 1 h at room temperature (RT). To examine DNA fragmentation using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, BL stage embryos were collected and fixed in 3.7% paraformaldehyde in PBS for 1 h at RT.

Confocal microscopy. To allow for double staining of actin, microtubules and DNA, oocytes were fixed in 4% paraformaldehyde in PBS for 30 min, at room temperature, and then transferred to a membrane permeabilization solution (0.5% Triton X-100) for 20 min. After 1 h in blocking buffer (1% BSA-supplemented PBS), oocytes were incubated overnight at 4°C or for 4 h at room temperature with 1:200 anti-α-tubulin-FITC (Sigma) or 10 mg/ml Phalloidin-FITC (Sigma). After three washes in a washing buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), the oocytes were labeled with 1:100 Alexa Fluor 488 goat-anti-mouse IgG (for microtubules staining) for 1 h, at room temperature. The samples were co-stained with Hoechst 33342 for 10 min and were then washed three times in washing buffer. The samples were mounted on glass slides and examined with a confocal laser-scanning microscope (Zeiss LSM 710 META, Germany). At least 30 oocytes were examined for each group.

Western blot analysis. Embryos were thawed at room temperature and 25 μl Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) were added. The samples were heated at 95°C for 5 min, and the samples were separated by electrophoresis through a Mini-PROTEAN TGXTM Precast Gel (Bio-Rad) for 2 h at 100 V. Proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane (iBlotTM Gel Transfer Stacks; Invitrogen, Grand Island, NY, USA). After blocking with 5% bovine serum albumin (BSA) in tris-buffered saline (TBS) for 1 h, the membrane was incubated with a c-PARP antibody (Cell Signaling Technology, San Diego, NY, USA) diluted at 1:1000 in blocking solution [20 mM Tris–HCl (pH 7.5), 250 mM NaCl, 0.1% Tween-20, 5% (w/v) BSA] for 1 h at room temperature. The c-PARP antibody recognizes the 89-kDa fragment of mouse PARP1 that results from caspase cleavage. After washing three times in TBST (20 mM Tris–HCl (pH 7.5), 250 mM NaCl, 0.1% Tween-20), the membrane was incubated for 1 h with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) diluted at 1:2000 in blocking solution at room temperature. After three 10-min washes in TBST, bands were visualized using an Enhanced Chemiluminescence (ECL) reagent (Animal Genetics Inc., Seoul, Korea).

Figure 1.
Figure 1.

Effect of doxorubicin on the survival of zygotes cultured in vitro. Cultures were exposed to different doses of doxorubicin and embryonic survival was evaluated on days 1, 2, 3 and 4 in culture. Values are represented as means±SEM (bar) of three independent experiments. As compared with the paired controls, *p<0.05.

Real-time RT-PCR with SYBR Green. To detect the expression of Bcl2l1, Bax and Casp3, mRNA was isolated from in vitro-cultured mouse embryos at different stages using a Dynabeads mRNA Direct Kit (Dynal Asa, Oslo, Norway). First-strand cDNA synthesis was achieved by reverse transcription of mRNA using an oligo(dT) primer and SuperScript II Reverse Transcriptase (Invitrogen Co.). The Bcl2l1, Bax and Casp3 primers are shown in Table I. Real-time PCR was performed in a final reaction volume of 20 μl in the presence of SYBR Green, a dye that fluoresces when bound to double-stranded DNA (qPCR kit; Finnzymes, Espoo, Finland). The PCR protocol was as follows: 10 min at 94 °C followed by 39 cycles of 94°C for 30 s, 60 °C for 30 s and 72°C for 25 s, and a final extension of 5 min at 72°C. Gene expression was normalized to mouse H2a mRNA and quantified using the 2−ΔΔCt method (21).

TUNEL assay BL stage embryos were fixed, washed in PBS/Polyvinylpyrrolidone (PVP) and permeabilized by incubation in 0.3% Triton X-100 for 1 h at RT. The embryos were washed twice in PBS/PVP and incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase enzyme (in situ Cell Death Detection Kit, Roche; Mannheim, Germany) in the dark for 1 h at 37°C. After being treated with 4 μg/ml RNaseA for 1 h at 37°C and counterstained with 40 μg/ml Hoechst 33342 to label all nuclei, the embryos were washed in PBS/PVP, mounted with slight coverslip compression and observed under confocal microscopy.

Statistical analysis. The general linear models (GLM) procedure in the SAS program (SAS institute Inc, NC, USA) was used to analyze the data from all experiments. Significant differences were determined using the Tukey's multiple range test and p<0.05 was considered significant.

Results

Effect of doxorubicin on the survival of early embryos. The effect of doxorubicin on the survival of embryos cultured in vitro is shown in Figure 1. The data are normalized to the starting number of zygotes in each treatment group. Cultures were exposed to different doses of doxorubicin on day 1, and embryo survival was evaluated on days 2, 3 and 4. Doxorubicin had no effect on zygote survival at a concentration of 5 nM. However, zygote survival at day 4 decreased significantly to 42%, 32% and 11% relative to controls after a 24-h exposure to 50 nM, 200 nM and 1000 nM doxorubicin, respectively.

Doxorubicin induces early DNA damage, fragmentation and inhibition of cell division. In previous studies, the effects of doxorubicin on mouse oocytes have been examined both in vivo and in vitro (3, 5, 6). Oocytes exposed to doxorubicin rapidly underwent morphological and biochemical changes consistent with apoptosis. In this study, doxorubicin induced fragmentation of zygotes after a 20-h exposure to 200 nM doxorubicin. DNA damage and inhibition of cell division were evident after a 20-h exposure to 50 nM doxorubicin (Figure 2). Our results confirmed that DNA damage and cytoplasmic fragmentation in zygotes are two independent events. A 1C (zygote) arrest that involved the disruption of nuclear DNA and changes in the actin and microtubule cytoskeleton was observed.

Figure 2.
Figure 2.

Doxorubicin induces cytoplasmic fragmentation and zygote arrest. A: Fragmented embryos: extensive cytoplasmic fragmentation was apparent after a 20-h treatment with 50 nM doxorubicin. B: Zygotes retained the normal morphology after a 20-h treatment with 50 nM doxorubicin. C,D: Zygotes showed abnormal morphology after a 20-h treatment with 50 nM doxorubicin. E: The control group reached the 2C stage after culture for 20 h in M16 (control medium). C-E: The nuclear status of denuded oocytes was determined by Hoechst 33342 staining (blue) and the cytoskeleton was examined by immunofluorescence of actin (red) and microtubules (green).

Effect of 5 nM doxorubicin on apoptotic gene expression during early embryonic development. The relative expression of apoptosis-related genes in early embryonic development was assessed after treatment with 5 nM doxorubicin in vitro (Figure 3). The expression of the apoptosis genes Casp3, Bax and Bcl2l1 was determined in several stages of mouse pre-implantation embryos using real-time RT-PCR (Figure 3A-D). Casp3 and Bcl2l1 mRNA levels were lower in 2C embryos compared to control, but Casp3 gradually increased from the 4C to the BL stage. Bcl2l1 decreased from the 2C to the MO stage, and then increased in the BL stage. In order to normalize RT-PCR reaction efficiency and to quantify the mRNA levels, samples were normalized against the internal reference H2a. There was no difference in the expression of Bax between the control and doxorubicin-treated groups. Therefore, doxorubicin affected the expression of the apoptosis-related genes Casp3 and Bcl2l1 during early embryo development, concomitant with the progression of apoptosis. Effect of 5 nM doxorubicin on PARP gene expression during early embryo development. The effect of doxorubicin on the expression of PARP1 and PARP2 was determined in several developmental stages of mouse pre-implantation embryos using real-time RT-PCR (Figure 3). PARP1 and PARP2 mRNA levels were higher in doxorubicin-treated embryos compared to controls, especially at the 2C and 4C stages (Figure 3A and B). At the MO and BL stages, PARP1 and PARP2 expression in treated embryos was similar to that of controls (Figure 3C and D). To normalize RT-PCR reaction efficiency and to quantify the mRNA levels, samples were normalized against the internal reference H2a. The data were consistent with that of caspase3 expression, indicating that PARP1 activity is involved in modulating caspase3. The data also suggested an interaction between PARP1 and PARP2.

Effect of 5 nM doxorubicin on the developmental competence of zygotes and on the number of cells in BLs. To assess the effect of doxorubicin on zygote developmental competence, we determined the rates of 2C, 4C, 8C, MO and BL stage formation. The developmental competence of the doxorubicin-treated group was significantly lower than that of the control group, at all developmental stages. By the BL stage, the rate was only 13% in the doxorubicin-treated group relative to controls (Figure 4). These data suggest that doxorubicin inhibited early embryo development by up-regulating apoptosis.

Figure 3.
Figure 3.

Relative expression of apoptosis-related genes (Bcl2l1, Bax and Casp3) and PARP family genes (PARP1 and PARP2) was determined in 2 cell (2C), 4 cell (4C), morula (MO) and blastocyst (BL) stage embryos treated with 5 nM doxorubicin. Messenger RNA levels of apoptosis-related genes were analyzed by real-time RT-PCR. *Significantly different from controls, p<0.05. Values are the mean±SEM of five separate experiments.

To further investigate the effect of doxorubicin on zygote developmental competence, the number of cells per BL and the apoptotic index (number of apoptotic cells/total number of cells) were measured (Figure 5). In the doxorubicin-treated group, the BL cell number was significantly lower and the apoptotic index was significantly higher relative to that of controls. These results confirmed that doxorubicin inhibited early embryo development by up-regulating apoptosis.

Effect of doxorubicin on c-PARP protein levels in early mouse embryos. To study the effect of doxorubicin on the PARP pathway, the level of c-PARP was measured by western blotting. The presence of the 89-kDa PARP1 cleavage fragment (c-PARP) confirmed that PARP1 was cleaved and that PARP1 activity was attenuated in the early mouse embryos. PARP1 inactivation in mouse embryos was dose-dependent, as indicated by the increasing levels of c-PARP as the dosage of doxorubicin increased. The highest level of c-PARP was detected after treatment with 200 nM doxorubicin, consistent with the greatest reduction in developmental competence at that dosage (see Figure 1).

Figure 4.
Figure 4.

Early embryonic development. The data show the percentage of embryos reaching the 2 cell (2C), 4 cell (4C), morula (MO) and blastocyst (BL) stages in control (white bars) and 5 nM doxorubicin-treated (black bars) embryos. Asterisks indicate a significant difference (p<0.01) relative to controls at the same stage. Values are the means±SEM of four separate experiments.

Discussion

Doxorubicin is known to have an injurious effect on oocytes (3, 5-6), but the effect of doxorubicin on early embryonic development are unknown. To determine the effects of different doxorubicin concentrations on embryos, survival was evaluated on days 1, 2, 3, 4 after exposure to this agent. Doxorubicin had no effect on embryo survival at a concentration of 5 nM. However, embryo survival significantly decreased, to 42% and 11% after 4 days of exposure to 50 nM and 1000 nM of doxorubicin, respectively. Cytoplasmic fragmentation was rapidly induced at 200 nM of doxorubicin, and zygotes were arrested at the 1C stage in response to 50 nM doxorubicin. At a concentration of 5 nM, doxorubicin inhibited early embryonic development, but a few embryos still reached the BL stage. Embryo survival decreased, and the degree of damage to the embryos increased, with increasing concentrations of doxorubicin. We concluded that doxorubicin inhibited early embryonic development by up-regulating apoptosis.

Apoptosis is a form of cell death that takes place under physiological conditions and plays a key role in biological processes such as embryonic development, tissue remodeling and renewal, and the regulation of cell populations (22). Cytoskeleton components (microfilaments, intermediate filaments, microtubules, and other cytoskeleton-related proteins) are disrupted by apoptosis. The cytoskeleton also modulates death receptor and death receptor-ligand concentrations during apoptosis (23). To examine the effect of doxorubicin on the cytoskeleton, we examined the microtubules and actin. In the treatment groups, cortical actin staining was reduced and disorganized compared to the control group. The nuclear DNA was fragmented and dispersed. Therefore, we concluded that doxorubicin arrested zygotes at the 1C stage by disruption of DNA and of the cytoskeleton.

Figure 5.
Figure 5.

Total cell numbers and apoptotic rates in blastocysts (BLs). In vitro development of zygotes in the BL stage was examined. The total cell number per BL was quantified and the apoptotic rate (number of apoptotic cells/total number of cells) was compared in control and 5 nM doxorubicin-treated embryos. Asterisks indicate a significant difference (p<0.01) from control values. Values are the means±SEM of four separate experiments.

Figure 6.
Figure 6.

Doxorubicin-induced PARP cleavage. Cultures were exposed to different doses of doxorubicin, as indicated. Western blots of 50 zygotes per lane are shown. The culture times and treatments are indicated at the top of the figure. The amount of c-PARP increased in a dose-dependent manner. The blot was subsequently reprobed for Gapdh, as a loading control.

Apoptosis is the main cause of primordial germ cell and oocyte degeneration in the developing fetal ovary. The Bcl2 family controls programmed cell death during embryogenesis and includes both anti-apoptotic and pro-apoptotic members. Bcl2l1 functions to protect against apoptosis (24-26). In contrast, another group of highly conserved genes, BAK and BAX, are positive regulators of apoptosis that lead to the release of cytochrome c and activation of the caspase cascade (27). In this study, we found that doxorubicin treatment changed the expression of Bcl2l1 and Casp3, but did not affect Bax. This result may reflect the ability of zygotes to suppress the apoptotic process at the molecular level. Zygotes were arrested in the 1C stage on treatment with 50 nM doxorubicin. This arrest may be due to the inhibition of DNA synthesis and induction of apoptosis. Bcl2l1 and Casp3 expression was up-regulated during embryogenesis in doxorubicin-exposed embryos. This result highlights theimportance of Bcl2l1 and Casp3 in early embryonic development.

Early embryonic development in the BL stage was examined, and some embryos did indeed survive to the BL stage upon treatment with 5 nM doxorubicin. PARP expression was examined since the deleterious effects of doxorubicin on DNA might affect DNA repair. PARP1 and PARP2 were both up-regulated in response to 5 nM doxorubicin, suggesting that the gene products may interact. PARP2 was recently shown to be critical for PARP1-mediated activation of homologous recombination at stalled replication forks (28).

During apoptosis, PARP1 cleavage by other enzymes prevents itself from activation and the resultant ATP depletion that would otherwise be triggered during DNA fragmentation (29-32). The high expression of PARP in the 5-nM doxorubicin treatment groups, especially in the 2C and 4C stages, is consistent with the low Casp3 expression levels in doxorubicin-treated 2C and 4C embryos. Taken together, these results suggest that doxorubicin inhibits early embryo development by causing DNA damage and blocking DNA repair mechanisms, which ultimately stimulates apoptosis.

This study examined the effects of doxorubicin in early embryos, mirroring the stages affected in vivo in female patients treated with chemotherapy. Our results indicated a dose-dependent sensitivity of the zygotes to doxorubicin, as indicated by increasing levels of apoptosis. Rapid disintegration of the chromosomes and a pronounced disruption of the cytoskeleton were detected in response to doxorubicin treatment. These findings reflect the potential clinical outcome for female patients undergoing cancer treatment. The current findings suggest that doxorubicin-induced apoptosis was the result of the disruption of DNA repair and fragmentation of the cytoskeleton.

Acknowledgements

This work was supported by a grant from the Next-Generation BioGreen 21 Program (no. PJ009098, PJ00832302, PJ008067 and PJ0090802012), Rural Development Administration, Republic of Korea.

  • Received March 4, 2012.
  • Revision received April 8, 2012.
  • Accepted April 12, 2012.

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Doxorubicin Induces Early Embryo Apoptosis by Inhibiting Poly(ADP ribose) Polymerase
QING-LING WANG, SHAO-CHEN SUN, JEMESBOND HAN, YONG-CHUL KWAK, NAM-HYUNG KIM, XIANG-SHUN CUI
In Vivo Sep 2012, 26 (5) 827-834;
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