Epigenetic programming in the preimplantation rat embryo is disrupted by chronic paternal cyclophosphamide exposure

Abstract

Preconceptional paternal exposure to cyclophosphamide, a widely used anticancer agent, leads to increases in embryo loss, malformations, and behavioral deficits in offspring; these abnormalities are transmissible to subsequent generations [Auroux, M., Dulioust, E., Selva, J. & Rince, P. (1990) Mutat. Res. 229, 189–200]. Little information exists on the mechanisms underlying this male-mediated developmental toxicity. We assessed the impact of paternal cyclophosphamide exposure on the dynamic regulation of histone H4 acetylation at lysine 5 and DNA methylation in preimplantation rat embryos. Zygotes sired by drug-treated males displayed advanced developmental progression, increased pronuclear areas, and disruption of the epigenetic programming of both parental genomes. Early postfertilization zygotic pronuclei were hyperacetylated; by mid-zygotic development, male pronuclei were dramatically hypomethylated, whereas female pronuclei were hypermethylated. Micronuclei were substantially elevated, and histone H4 acetylation at lysine 5 localization to the nuclear periphery was disrupted in two-cell embryos fertilized by cyclophosphamide-exposed spermatozoa. This finding demonstrates that paternal exposure to this drug induces aberrant epigenetic programming in early embryos. We hypothesize that disturbances in epigenetic programming contribute to heritable instabilities later in development, emphasizing the importance of epigenetic risk assessment after chemotherapy.

Long-term survival rates after childhood and reproductive-aged cancers are rising dramatically, eliciting concern for the health of offspring conceived after cytotoxic therapy (1). Of great concern, germ-line instabilities may be manifested in surviving offspring and be propagated across multiple generations. Paternally transmitted chromosomal aberrations are present in the mouse zygote after preconceptional exposure to six mutagens; the extent of abnormalities was suggested to predict the developmental competence of the embryo (2, 3). The first evidence of persistently elevated mutation rates in second generation progeny is after paternal exposure to irradiation in mice (4). A plausible mechanism by which toxicant perturbation of DNA may lead to heritable alterations in the genome is by epigenetic modifications. The impact of aberrant epigenetic reprogramming on embryonic development in cloned animals has recently received attention (5, 6). We report experimental evidence of epigenetic deregulation in the naturally fertilized zygote after paternal drug exposure.

Cyclophosphamide, an extensively used chemotherapeutic (1, 7) and immunosuppressive agent, targets rapidly dividing cells, alkylating DNA at the N7 position of guanine (7). Spermatogenesis is characterized by complex, sequential stages of differentiation, chromosomal assortment, and chromatin remodeling (8), rendering male germ cells highly susceptible to genotoxicants. Cyclophosphamide induces DNA–DNA and DNA–protein cross-links and DNA single-strand breaks (7), altering the unique structural organization of the paternal genome (9). Sperm chromatin remodeling is essential for imposing predetermined epigenetic programs and involves a wave of hyperacetylation, replacement of histones by sperm-specific protamines (10), nuclear condensation, and spermatid DNA remethylation (11), producing a transcriptionally inert spermatozoal genome. After fertilization, the paternal chromosomes decondense and undergo extensive remodeling in association with dramatic epigenetic changes that occur asymmetrically in both parental genomes to establish the coordinated parent-specific programs essential for embryogenesis (12–14).

Epigenetic disturbances that occur soon after fertilization, involving somatic and germ-line cells before lineage specification, may be particularly detrimental. The inheritance of epigenetic defects may lead to developmental and neurological abnormalities, aberrant disease phenotypes, and subtle changes in gene expression, resulting in an elevated predisposition to cancer in the offspring and subsequent generations (reviewed in refs. 15 and 16). We propose that the DNA damaging effects of paternal cyclophosphamide exposure disrupt the epigenome of male germ cells during this vulnerable state of chromatin remodeling.

Materials and Methods

Drug Treatment, in Vivo Rat Embryo Production, and Collection. Adult male (body weight, 350–400 g) and virgin female (body weight, 225–250 g) Sprague–Dawley rats were purchased from Charles River Canada (St. Constant, Quebec) and housed at the Animal Resources Centre, McIntyre Medical Building, McGill University. Animals received food and water ad libitum and were maintained on a 12-h light/12-h dark photoperiod. After one week of acclimatization, male rats were randomly assigned to one of two treatment groups (n = 10 per group) and gavaged with saline or 6 mg/kg per day of cyclophosphamide six times per week for 4–5 weeks (17–20). This treatment regime ensures the targeting of sperm chromatin organization and packaging during spermiogenesis (9).

During week 5 of treatment, each male was mated overnight with 2 control virgin females in proestrus. At 0900 hours the following morning, designated gestation day 0, pregnancies were confirmed; sperm-positive females were killed at 1300 hours on day 0 or at 1100 hours on day 1 to collect one-cell stage and two-cell stage embryos, respectively. Oviducts and proximal uteri were isolated, one-cell stage embryos were released into warm (37°C) 1% hyaluronidase (Sigma) for cumulus cell dissociation; two-cell embryos were flushed from the oviducts of pregnant dams with warm (37°C) M2 culture medium (Sigma). Embryos were prepared for immunofluorescent staining as described below. All animal protocols were conducted in compliance with the guidelines outlined in the Guide to the Care and Use of Experimental Animals, prepared by the Canadian Council on Animal Care.

Immunofluorescence. All embryo manipulations were done at room temperature unless otherwise stated. Embryos sired by saline and cyclophosphamide-exposed males were stained in parallel. Preimplantation embryos were washed in 1× PBS, pH 7.4 (Mg²⁺ and Ca²⁺ free), containing 1 mg/ml polyvinylpyrrolidone. Zonae pellucidae were removed by rinsing embryos in a drop of Acid Tyrode's solution. Methods used for indirect immunofluorescence were described by Santos and colleagues (5, 13). Embryos were fixed in 4% paraformaldehyde in PBS for 15 min, washed in 0.05% Tween 20 in PBS for 5 min, permeabilized for 30 min in a solution of 0.2% Triton X-100 in PBS, rewashed in 0.05% Tween 20 in PBS for 5 min, and blocked overnight at 4°C in 1% BSA and 0.05% Tween 20 in PBS. Immunofluorescence staining for DNA methylation patterns required additional treatment of all samples with 4 M HCL and 0.01% Triton X-100 for 20 min, followed by a 15-min neutralization of cells with 100 mM Tris·HCl, pH 8.5, subsequent to permeabilization.

To recognize the preferentially acetylated isoform of histone H4, embryos were incubated for 1 h in rabbit polyclonal anti-histone H4 acetylated at lysine 5 (H4-K5) (1:500 dilution; Abcam, Cambridge, MA) (14). They were washed vigorously in a series of fresh blocking solution (1 × 10 min, 1 × 30 min, and 1 × 10 min) and incubated for 1 h in Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (1:200 dilution; Molecular Probes); blocking solution washes were repeated. DNA was stained with 10 μg/ml DAPI for 30 min, embryos were washed in 0.05% Tween 20 in PBS for 10 min, and mounted in 3 μl of VectaShield antibleaching mounting medium (Vector Laboratories) on premarked slides.

DNA methylation was visualized with a mouse monoclonal anti-5-methylcytosine antibody (1:25 dilution) (5, 12, 13). Embryos were incubated for 1 h with the primary antibody, washed repeatedly in blocking solution, and incubated for 1 h with anti-mouse fluorescein conjugated-IgG secondary antibody (1:40 dilution; Amersham Pharmacia Biosciences). DNA was stained with 2 μg/ml propidium iodide (Molecular Probes) for 15 min, and embryos were mounted. Control experiments included the incubation of one-cell and two-cell stage embryos in secondary antibody only to confirm nuclear staining specificity. The second polar body serves as an internal staining control for 5-MeC because it does not undergo demethylation during preimplantation development.

Confocal Microscopy. Fluorescence was visualized by using a Zeiss LSM 510 Axiovert 100M confocal microscope equipped with a Plan-Apochromat ×63/1.4 oil DIC objective. The optimal conditions for laser scanning confocal microscope fluorescence imaging for each primary antibody were determined experimentally; identical brightness and contrast settings were used when collecting original images from both one- and two-cell embryos. All embryos were scanned at a speed of 7 with an optical slice of 0.7 μm, zoom factor equal to one and a pinhole setting of 96 μm. Sixteen scans of each optical section were compiled and averaged by the computer software (lsm 510, Zeiss) to give the final image that was 1,024 × 1,024 pixels in size. All embryos stained with anti-H4-K5 or anti-5-MeC, were imaged with a detector gain setting of 775 or 992, respectively. Images were collected and transferred into monochrome eight-bit TIFF files for quantitative analysis.

Quantitative Analysis. Quantitative analysis was done on single optical images of all embryos with the microcomputer imaging device mcid 7.0 (Imaging Research, St. Catherine's, ON, Canada) software. All threshold settings for intensity and saturation were maintained constant across all experimental groups. Approximately 100 embryos were examined for each preimplantation cleavage stage and for each treatment. The pronuclear and nuclear areas were measured as the total grain areas within the region of interest, consisting of chromatin regions with the exclusion of the early embryonic precursor nucleoli. Fluorescence intensity was expressed as the mean integrated value of all pixels contained within the sample outline as measured on a brightness scale of 0–1. Zygotes were qualitatively categorized into five pronuclear stages (PN) based on the interpronuclear distance of the paternal and maternal genomes in the cytoplasm and male pronuclear morphology. Morphological analysis consisted of embryo classification according to the appearance of nucleoli within the paternal pronucleus: 1 large nucleolus, 1 large nucleolus with few smaller nucleoli, or multiple small nucleoli, corresponding to PN1 and 2, PN3, and PN4 and 5, respectively (13, 14).

Statistical Analysis. Student's t tests or Mann–Whitney rank sum tests were used to compare the progression of zygotic development, total grain areas and fluorescence intensity of parental pronuclei and two-cell stage nuclei of embryos from control animals with those that were fertilized with spermatozoa chronically exposed to cyclophosphamide (P < 0.05). χ² analyses were used to compare the proportion of two-cell stage embryos with H4-K5 peripheral staining with or without micronuclei sired by saline or cyclophosphamide males (P < 0.05). Error bars represent the mean ± SEM. Statistical analyses were done by using the sigmastat 2.03 software package (SPSS, Chicago).

Results

Paternal Cyclophosphamide Exposure Disrupts Zygotic Development. The transmission of DNA damage incurred in the male genome during 4 weeks of cyclophosphamide administration (20) significantly disrupted the rate of zygotic development after in vivo fertilization (Fig. 1). The proportion of zygotes sired by males chronically exposed to cyclophosphamide was significantly decreased (P = 0.029) early postfertilization at PN1 and 2, whereas at PN3, there was an increasing trend, compared to corresponding controls. At PN4, the proportion of zygotes fertilized by cyclophosphamide-exposed spermatozoa was significantly increased (P = 0.039) compared with controls. Comparable numbers of zygotes sired by control and cyclophosphamide-exposed males were observed at PN5.

Fig. 1.

The progression of zygotic development is disrupted after chronic paternal cyclophosphamide exposure. Zygotes fertilized by drug-exposed spermatozoa displayed advanced progression to PN4. The number of embryos analyzed at each pronuclear stage was as follows. Saline, n = 49, 96, 40, and 15 for PN1 and 2, 3, 4, and 5, respectively. Cyclophosphamide, n = 19, 115, 68, and 14 for PN1 and 2, 3, 4, and 5, respectively. Open bars, saline sired zygotes; filled bars, cyclophosphamide sired zygotes; *, P < 0.05.

We further investigated pronuclear integrity in zygotes sired by cyclophosphamide-treated males by assessing the chromatin areas of parental pronuclei (Fig. 2). Immediately after fertilization, at PN1 and 2, pronuclear grain areas of embryos sired by cyclophosphamide-treated males were not different from controls. Interestingly, the areas of both paternal and maternal pronuclei were significantly enlarged at PN3 and 4 (P < 0.01), maintaining increased chromatin dispersion at PN5, compared with controls. Thus, fertilization by spermatozoa chronically exposed to a toxicant perturbed chromatin structure in both parental genomes, leading to excessive decompaction and aberrant developmental progression in the zygote.

Fig. 2.

Paternal and maternal pronuclei of zygotes from cyclophosphamide-treated males are dramatically increased in size. Zygotic chromatin was counterstained with propidium iodide to determine pronuclear area. The number of embryos analyzed at each pronuclear stage was as follows. Saline, n = 29, 47, 19, and 5 for PN1 and 2, 3, 4, and 5, respectively. Cyclophosphamide, n = 12, 51, 38, and 5 for PN1 and 2, 3, 4, and 5, respectively. Cross-hatched bars, saline male pronucleus; black bars, cyclophosphamide male pronucleus; open bars, saline female pronucleus; gray bars, cyclophosphamide female pronucleus. *, P < 0.01.

Aberrant Reprogramming in Parental Genomes After Paternal Cyclophosphamide Exposure. Paternal exposure to a chemotherapeutic agent before conception disrupts the temporal patterns of both H4-K5 (Fig. 3) and 5-MeC immunostaining (Fig. 4) in the rat zygote, indicating that genotoxic effects with an epigenetic basis are manifested very early after fertilization.

Fig. 3.

Histone H4 hyperacetylation in rat zygotes fertilized by cyclophosphamide-exposed spermatozoa. (A) Embryos were stained by indirect immunofluorescence by using an antibody to H4-K5 (green); DNA was counterstained with DAPI (blue). Acetylated histones assembled preferentially onto the paternal genome (a), became similar in both parental genomes (b and c), and remained constant for the duration of PN5 (d). In contrast, both parental genomes of zygotes sired by cyclophosphamide-treated males displayed increased H4-K5 staining soon after fertilization (e and f), returning to control levels later in development (g and h). (Scale bar: 20 μm.) (B) Quantitative analysis of H4-K5 fluorescence intensity. Paternal and maternal pronuclear histone H4 is significantly hyperacetylated in zygotes sired by cyclophosphamide-treated rats from PN1 to PN3. The number of embryos analyzed at each pronuclear stage was as follows: saline, n = 20, 49, 21, and 10 for PN1 and 2, 3, 4, and 5, respectively; cyclophosphamide, n = 7, 64, 30, and 9 for PN1 and 2, 3, 4, and 5, respectively. M, male pronucleus; F, female pronucleus, PB, polar body; crosshatched bars, saline male pronucleus; black bars, cyclophosphamide male pronucleus; white bars, saline female pronucleus, gray bars, cyclophosphamide female pronucleus. *, P < 0.05, **, P < 0.001.

Fig. 4.

Abnormal DNA methylation patterns in rat zygotes fertilized by cyclophosphamide-exposed spermatozoa. (A) Embryos were stained by indirect immunofluorescence by using an antibody to 5-MeC (green); DNA was counterstained with propidium iodide (red). Paternal pronuclei are dramatically hypomethylated in zygotes fertilized by drug-exposed spermatozoa (e–h) compared with controls (a–d) (Scale bar: 20 μm.) (B) Quantitative analysis of 5-MeC fluorescence intensity. In addition to the highly significant hypomethylation of the damaged paternal genome, maternal pronuclear 5-MeC immunofluorescence was significantly increased at PN3 in zygotes sired by cyclophosphamide-treated rats. The number of embryos analyzed at each pronuclear stage was as follows: Saline, n = 29, 47, 19, and 5 for PN1 and 2, 3, 4, and 5, respectively. Cyclophosphamide, n = 12, 51, 38, and 5 for PN1 and 2, 3, 4, and 5, respectively. M, male pronucleus; F, female pronucleus, PB, polar body; crosshatched saline male pronucleus; black bars, cyclophosphamide male pronucleus; white bars, saline female pronucleus, gray bars, cyclophosphamide female pronucleus. *, P < 0.05, **, P < 0.002. §, hypomethylation of paternal compared with maternal pronuclei, P < 0.001.

In the rat and mouse zygotes (14), paternal chromatin out-competes the maternal genome for the pool of acetylated histone H4 during PN2 (G₁) of the first cell cycle (Fig. 3Aa), whereas levels of pronuclear staining in both parental genomes become comparable by PN3 and PN4 (S phase) (Fig. 3A b and c) and remain constant for the duration of PN5 (G₂) (Fig. 3Ad). In zygotes fertilized by cyclophosphamide-exposed spermatozoa, both male and female pronuclei displayed enhanced levels of H4-K5 staining as early as PN2 (Fig. 3Ae); fluorescence continued to increase dramatically, becoming more intense at PN3 (Fig. 3Af) compared with corresponding controls. As zygotes progressed to PN4 and PN5 (Fig. 3A g and h), the pronuclei remained highly acetylated, similar to those observed in embryos sired by control spermatozoa (Fig. 3A c and d). Quantitative analysis of H4-K5 fluorescence intensity confirmed that male and female pronuclei in embryos sired by cyclophosphamide-treated males were significantly hyperacetylated beginning in G₁ (P < 0.05) and lasting into S phase (P < 0.001), corresponding to PN2 and PN3, respectively. In the later stages of zygotic development, the extent of H4 acetylation was not different between embryos sired by drug-exposed and control spermatozoa (Fig. 3B).

DNA methylation reprogramming, an epigenetic modification that is crucial for embryogenesis (5), was also markedly different in zygotes sired by cyclophosphamide-treated fathers (Fig. 4). Immediately after fertilization, both haploid pronuclei were equally methylated in zygotes sired by saline (Fig. 4Aa) and cyclophosphamide-treated males (Fig. 4Ae). In controls, male pronuclei underwent a gradual, active genomewide demethylation, whereas the female pronuclei remained hypermethylated with respect to the paternal genome (Fig. 4Ab–d). At PN5 (Fig. 4 Ad and B), the male pronuclei were significantly (P < 0.001) undermethylated compared with the female pronuclei. In contrast, the male pronuclei in zygotes fertilized by drug-exposed spermatozoa were dramatically hypomethylated at PN3 (Fig. 4Af) and remained globally undermethylated until the parental genomes were in close apposition at PN5 (Fig. 4A e–h).

Intriguingly, quantitative analysis of 5-MeC fluorescence intensity established that at PN3 (Fig. 4B), both parental pronuclei from embryos sired by cyclophosphamide-exposed spermatozoa were significantly affected (P < 0.002). The fluorescence intensities of male pronuclei were decreased, whereas those of the female pronuclei were aberrantly increased compared with controls. Male pronuclei from embryos sired by cyclophosphamide-treated fathers were consistently and significantly demethylated compared with controls through PN5 (Fig. 4B). These data emphasize the role of sperm chromatin composition in encoding parent-specific programs during preimplantation development (12).

Transmission of Aberrant Programming After the First Embryonic Cleavage. Genetic and epigenetic damage persisted to the two-cell stage (Fig. 5). Chronic cyclophosphamide treatment resulted in a marked induction in the proportion of two-cell stage embryos with micronuclei (Fig. 5A b and d) compared with controls (Fig. 5A a and c): 63% versus 4% (Fig. 5B; P < 0.001), respectively. Interestingly, embryonic micronuclei were marked by opposing epigenetic states; they were acetylated (Fig. 5Ab) and unmethylated (Fig. 5Ad). Similar chromatin abnormalities were not present in control embryos (Fig. 5A a and c).

Fig. 5.

Chromatin integrity and the localization of histone H4 acetylation at lysine 5 are disrupted in two-cell stage embryos sired by cyclophosphamide-exposed males. (A) Embryos were stained by indirect immunofluorescence by using an antibody either to H4-K5 (a and b) or to 5-MeC (c and d) (green); DNA was counterstained with DAPI (blue) or propidium iodide (red), respectively. Acetylation of histone H4-K5 was enhanced at the nuclear periphery of embryos sired by control males (a) but was widespread in embryos fertilized by cyclophosphamide-exposed spermatozoa (b). Two-cell stage embryos fertilized by cyclophosphamide-exposed spermatozoa contained micronuclei that were acetylated (b) and unmethylated (d). (Scale bar: 20 μm.) (B) The proportion of embryos with micronuclei was dramatically increased after chronic paternal cyclophosphamide exposure compared with controls. (C) Paternal cyclophosphamide exposure significantly decreased the proportion of embryos with a spatially restricted H4-K5 staining pattern in the two-cell stage subset without micronuclei. Saline: n = 88; cyclophosphamide: n = 26. Open bars, saline sired embryos; filled bars, cyclophosphamide sired embryos; arrows indicate micronuclei. *, P = 0.004, **, P < 0.001.

Paternal drug treatment also disrupted the spatial localization of histone H4 acetylation in two-cell embryos whether assessed collectively (data not shown) or as a subset excluding those embryos with micronuclei (Fig. 5A a and b). Acetylation of H4-K5 was enhanced at the nuclear periphery of embryos sired by control males (Fig. 5Aa); however, this spatially restricted staining pattern was often obliterated in embryos fertilized by drug-exposed spermatozoa (Fig. 5Ab). Analysis of the subset of embryos without micronuclei revealed that significantly fewer embryos sired by cyclophosphamide-exposed males portrayed the sequestration of H4-K5 immunofluorescence staining at the nuclear periphery (Fig. 5C; P = 0.004). In contrast, the pattern of 5-MeC staining was not spatially altered and immunofluorescence intensity was unchanged (data not shown) in two-cell embryos without micronuclei sired by cyclophosphamide-treated males (Fig. 5Ad) compared with controls (Fig. 5Ac).

Discussion

Delivery of highly organized genetic material in the sperm nucleus is required for competent participation of the paternal genome in embryo development (21); subtle structural disturbances in sperm chromatin are sufficient to impede normal embryogenesis (22). Rat spermatozoa from males chronically exposed to cyclophosphamide have normal fertility (17) but altered nuclear decondensation patterns in vitro, decreased sulfhydryl content (23), and advanced male pronuclear formation in hamster oocytes (19). In this study, the proportion of embryos sired by cyclophosphamide-treated males that were at PN1 and PN2 was significantly reduced; significantly more zygotes advanced to PN4 compared with controls. Alkylation of sperm DNA and/or nuclear proteins such as protamines may loosen chromatin structure and reduce nuclear compaction, thereby affecting the nuclear organization of mature spermatozoa. Accelerated pronuclear formation after in vitro fertilization of mouse oocytes was also observed after inhibition of poly (ADP-ribosyl)ation, a covalent modification involved in epigenetic remodeling (24).

Nuclear decondensation rate before pronuclear formation is directly related to disulphide bond content (25). Distorted chromatin packaging may lead to an unusually permissive conformation after fertilization, allowing the inopportune access of proteins with chromatin modifying potential. Intriguingly, fertilization by cyclophosphamide-exposed spermatozoa increased chromatin decompaction in both parental pronuclei (Fig. 2), emphasizing the unique interaction of two sets of chromosomes coexisting in the zygote as apparently separate entities (26). Destabilization of maternal chromatin architecture in response to the introduction of a damaged paternal genome suggests that common regulatory factors that control differential gene activity in the parental chromosomes may be affected.

Histone acetylation and DNA methylation constitute intricate regulatory mechanisms with essential roles in the DNA packaging and programming of epigenetic information during preimplantation development (6, 27). Our finding that preconceptional paternal exposure to a chemotherapeutic agent perturbs both H4-K5 (Fig. 3) and 5-MeC immunostaining (Fig. 4) in the rat zygote suggests that many fundamental cellular processes may be dysregulated.

Zygotes sired by drug-treated males were hyperacetylated very early after fertilization (Fig. 3). The possible consequences of hyperacetylation, as early as G₁ of the one-cell stage, are numerous because histone modifications are involved in the regulation of replication, transcription, and cell cycle progression (28). Histone acetylation is also required for DNA repair and genomic integrity (29), suggesting that the early zygote initiates a heightened damage response when attempting to repair genomic perturbations incurred during spermiogenesis.

Core histone acetylation is intimately associated with transcriptionally competent chromatin and, as predicted by the histone code hypothesis (28), signals for the recruitment of the specific protein complexes required to regulate zygotic gene expression. Studies in which histone deacetylase activity is inhibited demonstrate that chromatin-modifying proteins are active in the zygote as early as G₁ and that accurate modulation is critical for preimplantation development (14). In bovine, the onset of DNA replication in both pronuclei is directly regulated by the paternal genome; moreover, the first zygotic S phase plays a pivotal role in chromatin template organization for subsequent transcriptional activities (30). The histone H4 hyperacetylation induced as a result of paternal cyclophosphamide exposure may permit atypical relief of the repressed chromatin state and provide a window of opportunity for promiscuous binding and assembly of transcriptional complexes, leading to inappropriate patterns of gene expression. Histone acetylation is implicated in cell memory (14); therefore, the adverse consequences of abnormal patterns of histone H4 acetylation may be transmitted from one cell generation to the next, as well as to future progeny, thereby fitting the definition of an “epigenetic code” (31).

DNA methylation, an epigenetic mark associated with transcriptional repression and genomic stability (27), is also significantly altered in the male and female pronuclei of zygotes fathered by cyclophosphamide-exposed males.

The patterns of DNA methylation established during early embryogenesis serve as a global repression mechanism essential to regulate genomic structure and preserve stable epigenetic chromosomal states (27). Deficient reprogramming of DNA methylation in cloned bovine embryos is highly correlated with diminished developmental potential (6), whereas inappropriate gene repression is linked to numerous human diseases (reviewed in ref. 16). Thus, the greatly altered zygotic DNA methylation pattern observed after paternal cyclophosphamide treatment may contribute to the serious neurological deficits and developmental failure transmitted to three successive generations (18, 32).

The presence of a unique phenomenon, zygotic pronuclear cross talk, was revealed in embryos fertilized by spermatozoa chronically exposed to cyclophosphamide. Interestingly, the maternal pronucleus was transiently destabilized, specifically, hyperacetylated (Fig. 3) and hypermethylated (Fig. 4), as a result of damage to the spermatozoal genome. Paternal and maternal genomes have distinct roles after fertilization. The damage-free maternal genome has the ability to initiate a p53-dependent checkpoint in response to DNA damage delivered by irradiated spermatozoa (33). We provide evidence that fertilization by spermatozoa carrying drug-induced DNA damage leads to aberrant epigenetic reprogramming of both the paternal and maternal genomes. Deregulation of parental pronuclear methylation and nuclear hypermethylation after the first cleavage division may be responsible for the altered expression of imprinted genes (19) and contribute substantially to impaired postimplantation development.

Micronuclei in two-cell stage embryos are a sensitive measure of the clastogenic effects of paternal drug exposure (34). The genetic damage accumulated during spermatogenesis (9) cannot be fully repaired in the zygote and continues to be transmitted through successive cleavage stages. Previous studies (17) have shown that the current cyclophosphamide regimen does not affect preimplantation embryo survival but results in 80% postimplantation loss, comparable with the proportion of embryos presenting with micronuclei. The two-cell embryos without micronuclei had aberrant epigenetic markings, suggesting that there may be programming abnormalities in the absence of extensive chromosomal damage. A unique observation is that embryonic micronuclei are marked by opposing epigenetic states; specifically, acetylated (Fig. 5Ab) and unmethylated (Fig. 5Ad), suggesting a permissive chromatin conformation. Hypomethylation after exposure to 5-azacytidine is implicated in chromatin decondensation and micronucleus formation (35).

Peripheral enrichment of H4-K5 is transiently restricted to the two-cell embryo, correlating with a major burst of transcription during the onset of zygotic gene activation (36). The spatial localization of histone H4 acetylation is disrupted after paternal cyclophosphamide treatment, suggesting a dysregulation of cell cycle progression at the two-cell stage. It has been proposed that a replication-dependent mechanism moves chromatin from replication sites to the nuclear periphery; histones are further acetylated, gaining a permissive ground state for transcription factor binding and establishing a gene expression pattern compatible with continued development (37). Defects at the level of the transcriptional machinery and alteration of the gene activation program are evident in two-cell embryos sired by cyclophosphamide-treated males (19), supporting the hypothesis that the chromatin conformation necessary to temporarily localize acetylated histone H4 is disturbed.

Thus, cyclophosphamide-induced DNA damage in the paternal genome relays defective messages as early as the first round of DNA replication in the zygote, manifesting profound epigenetic changes in chromatin structure and function in a manner subtle enough to maintain fertility and perpetuate long-term genetic instabilities.