The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo
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Environmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation.
We performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 μg/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts.
We have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.
KeywordsTCDD Blastocyst Stage Aryl Hydrocarbon Receptor Inner Cell Mass Environmental Toxicant
The aryl hydrocarbon receptor (AhR) pathway is a widely expressed orphan receptor pathway activated by many environmental toxicants and carcinogens. AhR ligands, including dioxins and polychlorinated biphenyls, induce a spectrum of developmental and toxic responses by modifying gene expression, altering hormonal profiles and disrupting cell proliferation and differentiation . Epidemiological studies in adult human populations have linked dioxin exposure to defects in immune, neurological and reproductive function, as well as cancer [2, 3, 4]. There is now a growing concern that tissue growth and differentiation during fetal development may be especially sensitive to dioxins and dioxin-like compounds. For example, accidental exposure of human mothers to some AhR ligands has been correlated with delayed growth and development, a number of physical abnormalities, as well as intellectual and behavioral deficits in their children . Moreover, animal studies show embryonic lethality, teratogenesis, cleft palate, hydronephrosis and growth retardation among the many adverse effects observed following gestational exposure to AhR ligands [6, 7, 8]. While it is accepted that maternal exposure to AhR ligands during gestation is detrimental to the health of offspring, neither the mechanism of toxicity nor the exact stages of development affected by dioxins have been fully elucidated.
Past studies examining maternal dioxin exposure and subsequent fetal health have primarily focused on post-implantation embryogenesis, while the impact of environmental contaminants on the peri-conceptional and pre-implantation period remained largely unexplored. The maternal environment during this earliest window of development has been hypothesized as critical to the long term health of offspring . For example, poor maternal nutrition around the time of conception and during pre-implantation development reduces birth weight in humans  and animals  and predisposes offspring to hypertension later in life . Similarly, peri-conceptional exposure of mice to environmentally relevant doses of the environmental estrogen bisphenol A induces errors in meiotic chromosome segregation, yielding embryos that survive gestation but give rise to offspring with frank genetic deficits . Therefore, toxic exposure during pre-implantation embryonic development could potentially induce long term effects on fetal and offspring health.
Compacting morulae may be particularly vulnerable to the effects of environmental toxicants. Compaction is a morphogenetic process during which mammalian embryos undergo major cytoplasmic, nuclear and cytoskeletal remodeling events that lead to the establishment of apical-basal polarity [13, 14, 15, 16, 17]. Polarization permits the differentiative divisions that lead to the allocation of trophectoderm (TE) and the inner cell mass (ICM) for the placental and embryonic lineages, respectively . Therefore, disruption of pre-implantation embryo morphogenesis by AhR ligands could conceivably have consequences for lineage allocation. This concept is supported by the finding that in vitro exposure of 2 cell mouse embryos to TCDD results in increased cavitation rates, a functional measure of TE differentiation . Similarly, Tsutsumi et al. showed that in vitro exposure of 2 cell mouse pre-implantation embryos to very low levels of TCDD reduced the number of pre-implantation embryos that developed to 8 cells relative to controls, whereas blastocyst formation of the surviving 8 cell pre-implantation embryos was accelerated . These studies suggest differential and stage specific effects of TCDD during pre-implantation development.
The extent to which TCDD, at environmentally relevant doses, perturbs pre-implantation mammalian development in intact reproductively fit animals has yet to be fully evaluated. Thus, in a well-established rat model, we studied the effect of maternal TCDD exposure on early embryogenesis with respect to blastomere nuclear and cyto-architecture. We show specific nuclear and cytoskeletal modifications revealed from a systematic 3D confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to TCDD. Both chronic and acute maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping, aberrant cytokinesis and distortion of nuclear shape and position. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts. These studies raise further concerns regarding the consequences of early embryo exposures to prevalent environmental toxicants like TCDD.
Chronic maternal TCDD exposure disrupts 8–16 cell pre-implantation embryo morphogenesis
Numbers and health status of pre-implantation embryos collected following chronic and acute maternal TCDD exposure
Treatment (# animals)
Average # embryos/animal (range)
Average # blastomere/embryo (range)
# Normal embryos/total embryos (%)
Chronic exposure (compaction)
50 ng/kg/wk TCDD (3)
Acute exposure (compaction)
50 ng/kg TCDD (5)
1 μg/kg TCDD (6)
Acute exposure (blastocyst)
50 ng/kg TCDD (3)
1 μg/kg TCDD (5)
Acute periconceptional TCDD exposure disrupts 8–16 cell pre-implantation embryo morphogenesis
These initial findings prompted further analysis of cytoskeletal and nuclear characteristics in pre-implantation embryos to determine if limiting TCDD exposure to the time between oocyte maturation, ovulation and implantation (i.e. the periconceptional period) would similarly modify pre-implantation embryo organization. Mature naturally cycling female rats were exposed to a single dose of TCDD (50 ng/kg or 1 μg/kg) or vehicle on the evening of proestrus and compaction (8–16 cell) and early blastocyst stage (32 cell or more) pre-implantation embryos were collected and analyzed.
Acute periconceptional TCDD exposure permits pre-implantation embryo survival to blastocyst
We then asked whether the striking modifications evident at compaction were propagated through to the early blastocyst stage. In particular, it seemed likely that the occurrence of monopolar spindles would abrogate efficient cell cycle progression and negatively impact further pre-implantation embryo development. However, exposure to TCDD affected neither the number of pre-implantation embryos surviving to blastocyst nor the average number of cells within each blastocyst (Table 1), suggesting a normal rate of cell cycle progression in treated pre-implantation embryos.
Exposure to environmental toxicants, such as TCDD, prior to and during the earliest stages of pregnancy has been linked to developmental disabilities after birth in both human and animal studies [5, 6, 7, 8]. We conducted a systematic high resolution confocal microscopy analysis of rat pre-implantation embryos that has revealed previously unappreciated morphogenetic defects following maternal TCDD exposure. We have shown that both chronic and acute maternal exposure to TCDD induced nuclear and cytoskeletal defects in pre-implantation embryo morphogenesis. Specifically, our studies revealed effects of TCDD on 1) mitotic spindle integrity, 2) chromosome alignment, 3) nuclear and cellular size and shape and 4) cytokinesis efficiency. Furthermore, we have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival and development to the blastocyst stage is not compromised. To the best of our knowledge, this is the first in vivo study to demonstrate a critical window of early mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.
The comparison of acute verses chronic maternal exposure to TCDD on subsequent embryo quality in the current study encompasses examination of both dose effects and transgenerational toxicant actions. Dams in the acute exposure paradigm were exposed to low and high doses of TCDD limited to a single administration immediately preceding ovulation. The half life of TCDD in the rat is approximately 3 weeks, implying significant exposure until collection of the embryo. In the chronic exposure model, continuous exposure to TCDD in the dam began in utero and continued until breeding sacrifice at 3 months of age. In other words, the acute exposures were comprised of exposures to both the mother and the embryo, while the chronic exposure entails exposure of grandmother, mother and offspring. The results presented here show that both acute and chronic TCDD treatment protocols significantly compromised embryo quality.
It is likely that pre-implantation embryos are a direct target for TCDD, given that AhR is expressed throughout pre-impanation development  and that in vitro exposure of mouse pre-implantation embryos to TCDD accelerates differentiation of the blastocyst [19, 20]. However, due to the in vivo experimental design employed in this study, we can not rule out effects of TCDD on the oocyte nor on the mother's physiology, as factors contributing to the outcomes realized during pre-implantation embryo development. AhR ligands are known endocrine disruptors  and have also been shown to compromise oocyte quality by inducing apoptosis in cumulus cells . Additionally, recent studies have suggested that TCDD may actually accumulate in the follicular fluid and in the uterus, emphasizing the importance of the mother's physiology in contributing to pre-implantation embryo health and the necessity for in vivo studies [20, 25].
One of the most striking effects of TCDD, at both doses, was the induction of aberrant mitotic spindles and a failure in chromosome alignment in compaction stage pre-implantation embryos. AhR ligands, such as TCDD, may be involved in generating meiotic spindle aberrations by causing local increases in the concentration of 2-methoxyestradiol (2-ME) . 2-ME binds tubulin and influences microtubule polymerization and function. It is an endogenous metabolite of 17β-estradiol normally present within granulosa cells and follicular fluid of the ovary. Exposure to elevated levels of 2-ME causes spindle abnormalities, chromosome congression failure and nondisjunction in mouse oocytes and it is conceivable that such abnormalities would be further propagated within the developing embryo. Interestingly, exposure of bovine pre-implantation embryos to 2-ME does not inhibit passage from morula to blastocyst, and the cell cycle proceeds despite aberrations in spindle morphology .
Similarly, exposure to TCDD did not inhibit development to blastocyst and the data presented here clearly demonstrate that the overall structure and morphology of treated blastocysts were similar to control blastocysts. However, the widespread prevalence of defects uncovered in compaction stage pre-implantation embryos of treated animals, together with the relative paucity of cells exhibiting defects at the blastocyst stage, is disconcerting. Thus, a central question raised by this work is "What is the fate of aberrant compaction stage blastomeres?" We suggest three scenarios that may contribute to the survivability of TCDD exposed pre-implantation embryos. Firstly, defective cells may be eliminated by apoptosis during the transition from compacted pre-implantation embryo to blastocyst. Brison and Shultz showed apoptosis only occurs after compaction and is predominantly located in the ICM of the mouse . However, TCDD exposure did not increase the incidence of pyknosis in pre-implantation embryos in this study and thus elimination of TCDD-induced defects by selective apoptosis seems unlikely. This is consistent with an earlier study in which in vitro exposure of 2, 4, or 8 cell mouse pre-implantation embryos to TCDD did not significantly increase the number of TUNEL positive cells, alter the Bax/Bcl-2 expression ratio, or change cell number at the blastocyst stage .
Alternatively, defective cells may initiate repair mechanisms to rectify errors. Surveillance mechanisms that alert cells to impending errors in chromosome segregation exist in many normal somatic cells exhibiting a stable euploid condition. Such cell cycle checkpoints are engaged in response to structural aberrations as wide-ranging as chromosome misalignment to centrosome number . Notably, monopolar spindles with poorly aligned chromosomes are defects likely to activate cell cycle checkpoints. However, knockout and transgenic studies suggest that checkpoint controls may not be operational until the time of implantation in mouse embryos . Furthermore, lack of checkpoint activity in human embryos is suggested by the extreme degree of aneuploidy and mixoploidy seen in association with defects in mitotic spindle organization [31, 32]. If cell cycle checkpoints were activated in rat pre-implantation embryos, one might expect either an increase in the mitotic index or decrease in total cell number to be evident in blastocysts derived from TCDD exposed animals compared to controls. While there was no obvious increase in the presence of mitotic figures or cell number, further studies will be required to fully investigate this possibility.
It is possible that our detection of monopolar spindles was biased by a prolonged prometaphase delay that yielded euploid daughter cells after a correction that we were unable to detect in fixed samples. While this seems unlikely given the similar distribution of M-phase stages observed in control and treated pre-implantation embryos, on-going live cell recording experiments should resolve this dilemma. Moreover, cytogenetic assays will be needed to establish the incidence of aneuploidy, known to be elevated in human embryos exhibiting similar spindle defects, to better understand the status of checkpoint controls during this critical juncture during mammalian embryogenesis.
A final possibility is retention of defective blastomeres and their contribution to either or both of the lineages established in the blastocyst. While it is interesting to speculate that the TE lineage naturally undergoes ploidy variations indicative of less stringent cell cycle checkpoint surveillance , our data to date cannot rigorously assign fates to the aberrant blastomeres present during compaction. Given the changes in nuclear shape and the cytoskeleton resulting from exposure to TCDD during oocyte maturation and pre-implantation development, the emergence of cell polarity and lineage assignment during the compaction process may be impacted in subtle ways and yet still have major consequences for the later stages of embryogenesis. For example, the disruption of nuclear architecture is the central factor underlying a physiologically diverse group of inherited diseases known as laminopathies . Moreover, the epigenetic assignment of blastomeres to TE or ICM immediately precede the time we report here to be most sensitive to the effects of TCDD . In this regard, Skinner and colleagues have recently demonstrated altered epigenetic marks following exposure to environmental estrogens leading to disease phenotypes in adult male offspring that were passed on to subsequent generations . It was also recently shown that in vitro exposure of mouse pre-implantation embryos to TCDD increased methyl transferase activity, altered the methylation status of imprinted genes H19 and Igf2 and retarded subsequent fetal growth . Thus, disruption of epigenetic programming made during compaction provides a plausible mechanism by which pre-implantation exposure to TCDD could affect later development. In this light, the persistence of nuclear defects in shape and position may be subtle indicators of disruption of chromatin remodeling and epigenetic reprogramming during oocyte maturation and development up to compaction.
We have observed that compaction stage pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. The present work assumes particular relevance when considered together with recent evidence suggesting long term impacts on the health and well-being of offspring following environmental perturbations during the periconceptional and pre-implantation period [11, 38]. While the pre-implantation embryo may exhibit resiliency and plasticity in obtaining a state of implantation competence, perturbations during this critical window of development may be propagated with further cellular expansion and could abrogate developmental processes that will only surface after birth. Identifying the specific targets of TCDD in the pre-implantation embryo, especially those that link cell cycle control with cytoskeletal and nuclear remodeling, represents an important avenue for future investigation.
Female Sprague-Dawley rats (Charles River Laboratories) were housed under a 12L:12D photoperiod at an ambient temperature of 23 ± 2°C, with food and water ad libitum. All procedures were approved by the University of Kansas Medical Center Institutional Animal Care and Use Committee. In all experiments, pre-implantation embryos were obtained from naturally mated rats. Estrus cycles were monitored by vaginal cytology, with normal estrous cycle duration of 4–5 days . Source and purity of TCDD: CAS 1746-01-6; MW, 321.9; purity, > 99%.
In experiment 1, rats were exposed chronically to doses of TCDD that mimic exposure of high risk populations in humans [40, 41]. Weekly oral dosing was used in a previously validated regime [21, 42]. Initially, pregnant dams (n = 3 for each experimental group) received an oral dosing of TCDD (50 ng/kg) or corn oil vehicle (4 ml/kg) on day 14 and 21 of gestation and then on days 7 and 14 postpartum to provide in utero and postnatal lactational exposure, respectively. On postnatal day 21, female pups (n = 3 per experimental group) were weaned and orally dosed with TCDD (50 ng/kg) or corn oil vehicle (4 ml/kg), with dosing continued at weekly intervals thereafter. At 3 months of age, proven males were introduced on the evening of proestrus and mating was confirmed by the presence of sperm on vaginal cytology the following morning. Pre-implantation embryos were collected in FHM (Chemicon) media pre-warmed to 37°C by flushing oviducts and uteri on day 4.5 post coitum.
In experiment 2, female Sprague-Dawley rats (n = 3–6 for each experimental group) received a single oral dose (50 ng/kg or 1 μg/kg) of TCDD or corn oil vehicle (4 ml/kg) on the evening of proestrus and were housed with males of proven fertility. At the time of dosing rats were 50 days of age. Again, mating was confirmed by the presence of sperm on vaginal cytology the following morning. Pre-implantation embryos were collected in FHM (Chemicon) media pre-warmed to 37°C by flushing oviducts and uteri on day 4.5 or 5.5 post mating.
Pre-implantation embryos were processed for microtubule, DNA and f-actin immunofluorescence as previously described . Immediately following their collection, pre-implantation embryos were fixed for 30 min in 4% PFA at 37°C and stored at 4°C in wash solution comprising PBS supplemented with 2% BSA, 2% skim milk powder, 2% normal goat serum, 100 mM glycine, 0.01% Triton-X-100 and 0.2% sodium azide until processing for immunofluorescence. Pre-implantation embryos were extracted for 30 min at room temperature in 0.1% Triton-X-100 and incubated overnight at 4°C in wash solution. For immunostaining of microtubules, embryos were first incubated with mouse monoclonal anti-αβ tubulin (Sigma) diluted 1:100 in wash solution for 1 h at 37°C, followed by Alexa 488 labeled goat anti-mouse IgG (Molecular Probes) diluted 1:1000 in wash solution for 1 h at 37°C. DNA was stained with Hoechst 33258 (1 μg/ml in wash solution) for 30 min and f-actin integrity was analyzed by staining with rhodamine labeled phalloidin (1 μg/ml in wash solution; Molecular Probes) for 30 min. Pre-implantation embryos were mounted under cover slips without compression in medium containing 50% glycerol and 25 mg/ml sodium azide.
Pre-implantation embryos were analyzed on a Zeiss LSM Pascal confocal imaging system mounted on a Zeiss Axioscope II using UV (405 nm), HeNe (543 nm) and Argon (488 nm) laser excitation. For every embryo, a complete Z-axis data set was collected at 0.8 μm intervals (~50 sections/embryo) using a x63 oil objective (na = 1.4). Laser power, gain and offset settings were not changed during acquisition. Line scans and spatial restoration and 3 dimensional projections for each Z-series data set were computed and analyzed using Zeiss LSM 5 Image Browser.
Classification of pre-implantation embryos
Pre-implantation embryos were classified as abnormal if they contained blastomeres exhibiting one or more of the following: irregular size, irregular shape, weak or undetectable f-actin or tubulin, cellular fragmentation or micronuclei. Additionally, blastomeres containing metaphase-like chromosomes with mitotic spindles absent, or deviating from a focused bipolar microtubule array, were considered abnormal.
Chi-square was used to analyze the proportion of normal and abnormal pre-implantation embryos. P-values of less than 0.05 were considered significant.
We wish to thanks our colleagues Drs. Renata Ciereszko and Kelli Valdez for their critical reading of this manuscript. This research was supported by NIH/NIEHS-012916 (BKP), ESHE Fund (DFA), Hall Family Foundation (DFA and KJH) and Biomedical Research Training Grant KUMC (KJH).
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