Post-implantation Embryo Culture Research Articles

A hexa-species transcriptome atlas of mammalian embryogenesis delineates metabolic regulation across three different implantation modes

Mammalian embryogenesis relies on glycolysis and oxidative phosphorylation to balance the generation of biomass with energy production. However, the dynamics of metabolic regulation in the postimplantation embryo in vivo have remained elusive due to the inaccessibility of the implanted conceptus for biochemical studies. To address this issue, we compiled single-cell embryo profiling data in six mammalian species and determined their metabolic dynamics through glycolysis and oxidative phosphorylation associated gene expression. Strikingly, we identify a conserved switch from bivalent respiration in the late blastocyst towards a glycolytic metabolism in early gastrulation stages across species, which is independent of embryo implantation. Extraembryonic lineages followed the dynamics of the embryonic lineage, except visceral endoderm. Finally, we demonstrate that in vitro primate embryo culture substantially impacts metabolic gene regulation by comparison to in vivo samples. Our work reveals a conserved metabolic programme despite different implantation modes and highlights the need to optimise postimplantation embryo culture protocols.

Modified Xenopus laevis approach (R-FETAX) as an alternative test for the evaluation of foetal valproate spectrum disorder

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA.

Ex utero mouse embryogenesis from pre-gastrulation to late organogenesis.

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesisin mammals.

A serum-free and defined medium for the culture of mammalian postimplantation embryos

Whole embryo culture (WEC) of postimplantation rodent embryos is widely used for the study of mammalian embryogenesis and developmental toxicity testing. Its major advantage is that it allows direct access to embryos for experimental manipulations and the monitoring of their consequences that would otherwise not be possible or technically difficult to perform in utero. However, a major drawback of mammalian WEC is that the culture media currently in use display batch variations and are undefined, as they contain serum or serum replacements of unknown composition. Moreover, these media possess cell-signalling activities important for embryogenesis. Therefore, reproducibility of mammalian postimplantation WEC results may be affected by batch variation and their interpretation is complicated because the experimenter is unsure whether the embryo response to experimental perturbations is solely due to their action, or modified as a result of influences from undefined substances/signaling activities present in culture media. To alleviate these problems we investigated whether N2B27, a serum-free and defined medium, can support the in vitro development of postimplantation mammalian embryos. We show that N2B27 allows pre-gastrulation mouse embryos isolated at embryonic day 5.5 to develop to advanced gastrulation, reaching the mid- and late primitive streak stages. This is the first demonstration that postimplantation mammalian embryos can develop in vitro in a defined medium in the absence of serum and provides a novel WEC system for studying developmental mechanisms and testing for developmental toxicity during the early postimplantation period.

Evaluation of embryotoxicity of misoprostol using the whole embryo culture assay

Approximately 15% of misoprostol-induced-abortions may not be successful, leading to in utero exposure to the drug and to the induction of a series of defects including central nervous system, limb and visceral defects. A common proposal is that the drug causes disruption of the fetal vasculature leading to embryonic or fetal hypoxia. To evaluate the teratogenicity of misoprostol using the rat post-implantation embryo culture. Rat embryos were collected at the beginning of organogenesis and cultured in rat serum containing misoprostol at concentrations of 200, 2,000 or 20,000 pg/ml. Functionality, morphology and morphometry parameters were evaluated. Misoprostol induced a dose-dependent embryotoxic effect causing a decrease in embryo viability and function (poor vascular development and survival) and morphometry (alterations in branchial arches, heart and cephalic portions of the neural tube, among others). All the manifestations observed are indicative of the ability of misoprostol to directly induce developmental retardation and alterations.

Use of the rat postimplantation embryo culture to assess the embryotoxic potency within a chemical category and to identify toxic metabolites

The implementation of in vitro alternatives in the safety evaluation of chemicals in the animal intensive area of reproductive toxicity testing is highly desirable, but has been limited by issues around predictivity and applicability domains. The validation of alternatives may gain from a category approach, in which, rather than validating a test for the universe of chemicals, its predictive value is assessed for each class of chemicals for which the test represents relevant end point(s). We studied the embryotoxicity in rodent postimplantation whole embryo culture (WEC) of a series of phthalates and their metabolites. Phthalate diesters are widely applied industrial chemicals, their monoester derivatives being considered as their embryotoxic metabolites. The relative in vitro potency of three out of four monophthalates was found to mimick that of corresponding diphthalates tested in vivo. The phthalate that deviated from this ranking, monoethylhexylphthalate (MEHP), showed a relatively high in vitro toxicity as compared to in vivo data. This deviation could be explained through kinetic differences among phthalates, as shown between MEHP and monobutylphthalate. In addition, in vitro testing of specific secondary MEHP metabolites showed that they were all less potent than MEHP. This finding confirmed that MEHP in vitro embryotoxicity is most likely the best correlate to DEHP in vivo embryotoxicity. This study shows that a category approach in the assessment of the validation of in vitro alternatives is feasible, and can be improved when kinetic considerations are taken into account.

Application of a metabolizing system as an adjunct to the rat whole embryo culture

Rodent post-implantation whole embryo culture (WEC) is a validated and widely used method in both mechanistic studies and as a screening test for developmental toxicants. Since metabolism is lacking in the WEC because of the developmental stage of the embryo, pro-teratogenic compounds are not detected. We investigated the inclusion of an in vitro metabolizing system as a pre-incubation step in the existing WEC. We started by testing cyclophosphamide, a known pro-teratogen. Without metabolic activation, cyclophosphamide is not teratogenic to rat embryos, as evidenced by a lack of effect on either embryonic growth or on morphological development. After pre-incubation with Aroclor-induced hepatic microsomes, cyclophosphamide became highly embryotoxic in the WEC. We tested five additional compounds to prevalidate this method: valpromide, 2-acetylaminofluorene, 2-methoxyethanol, retinol, and benzo[a]pyrene. Results revealed that not all of these five compounds underwent (complete) metabolic activation. This is probably due to the fact that microsomes do not contain the full spectrum of hepatic enzymes. Attempts have been made to replace microsomes by S9 liver fractions, which do contain microsomal enzymes as well as a series of cytoplasmic enzymes. However, S9 appeared to be extremely toxic in the WEC. Future experiments have to be performed to explore alternative ways of complete metabolic activation.

Static Culture of Postimplantation Embryos for Imaging

Static Culture of Postimplantation Embryos for Imaging

Neurotoxic Effects of Alcohol and Acetaldehyde During Embryonic Development

Alcohol drinking during pregnancy results in abnormal fetal development, including fetal alcohol syndrome (FAS) in humans and experimental animals. FAS is characterized by two major effects, including central nervous system (CNS) dysfunction and multiple anomalies recognizable mainly as a typical face. However, the mechanisms of alcohol-induced embryotoxicity have not been clearly demonstrated. The aim of the present study was to investigate the possible mechanisms underlying ethanol-induced FAS in the developing embryo. First, ethanol-induced developmental abnormalities were investigated in vitro. Postimplantation embryos at gestation day (GD) 9.5 were cultured for 48 h and observed for morphological changes. Ethanol-mediated changes in proteins regulated apoptosis (p53 and bcl-2), antioxidant (vitamin E and catalase) activities, generation of reactive oxygen species (ROS), and oxidative DNA damage shown as 8-hydroxy-2′-deoxyguanosine (8-OHdG) were measured in embryonic midbrain cells. Alcohol or acetaldehyde significantly induced cytotoxicity in cultured rat embryonic midbrain cells. The levels of p53, bcl-2, and 8-OHdG were concomitantly changed by alcohol and acetaldehyde treatment in midbrain cells. Injured cells induced by ROS were increased by alcohol or acetaldehyde treatment in midbrain cells. Cotreatment with alcohol or acetaldehyde and catalase decreased cytotoxicity in midbrain cells. In postimplantation embryo culture, alcohol or acetaldehyde-treated embryos showed retardation of embryonic growth and development in a concentration-dependent manner. These results indicate that alcohol and its metabolite acetaldehyde induce fetal developmental abnormalities by disrupting cellular differentiation and growth. Data demonstrate that some antioxidants can partially protect against the alcohol-induced embryonic developmental toxicity.

Apparent lability of neural tube closure in laboratory animals and humans.

Neural tube defects (NTDs), a set of structural abnormalities affecting the brain, spinal cord, and the skeletal and connective tissues that protect them, are common malformations among humans and laboratory animals. The embryogenesis of the neural tube is presented to convey the complexity of the phenomenon, the multiplicity of requisite cellular and subcellular processes, and the precise timing of events that must occur for successful neural tube development. Interruption, even transitory, of any of these intricate processes or disruption of an embryo's developmental schedule can lead to an NTD. The population distribution of human NTDs demonstrates that genetic predisposition functions in susceptibility to NTDs. Data from animal studies support these concepts. NTDs are common outcomes in developmental toxicity safety assessments, occurring among control and treated groups. Numerous agents have caused increased levels of NTDs in laboratory animals, and species with shorter gestational periods appear more prone to toxicant-induced NTDs than those with longer gestations. Data from post-implantation whole embryo culture, although not predictive of human risk, are useful in studying neurulation mechanisms and in demonstrating the importance of maintaining embryonic schedules of development. We conclude that the concept that NTDs are produced by only a few toxicants that selectively target the developing nervous system is untenable. Rather, the combination of the time in gestation that an agent is applied, its dose, and its ability to disrupt critical processes in neurulation leads to NTDs. We further conclude that, because of both the relatively high prevalence and the multifactorial nature of NTDs, the mere occurrence of an NTD is insufficient for inferring that the defect was caused by an exogenous agent.

Embryotoxicity of carbamazepine in rat postimplantation embryo culture after in vitro exposure via three different routes

Postimplantation rat embryo culture is used widely for studies of embryotoxic effects on the isolated embryo after in vitro exposure to xenobiotic compounds. In this study, the relevance of three routes of exposure of the embryo in vitro was evaluated using the embryotoxic anticonvulsant carbamazepine. Embryotoxic effects were assessed, and analyses in conceptus tissues were done to reveal uptake and metabolism of the compound. Exposure via the culture medium resulted in neural tube defects and general retardation of growth and development. After injections into the amniotic or exocoelomic space, local membrane adhesions were found. Intra-amniotic exposure caused adhesions of the amniotic membrane with the embryonic neural plate, resulting in trapping of the membrane in the closing neural tube, as well as in open neural tube defects occurring in various areas of the neural tube. Only after exposure via the culture medium were amounts of carbamazepine detectable in the embryonic: tissue, correlating with the systemic effects found. It is concluded that uptake from the culture medium via the yolk sac circulation is the relevant exposure route to be used for embryotoxicity effect assessment.

Postimplantation mouse embryos cultured in vitro. Assessment with whole-mount immunostaining and in situ hybridization.

The postimplantation embryos of rodents have been particularly convenient to study in culture using the whole embryo culture (WEC) system developed by New. Two serious limitations of the method will be illustrated in the present paper and proposals will be made to improve the quality of the information. The first limitation is that the developmental period amenable to culture has not been significantly extended in recent years. In the present paper, we show that the culture of mouse presomitic stages for 48 h leads to poorly reproducible results and frequent dysmorphogenic embryos. We also show that early somite stages cultured for 54 h or less have a normal growth and differentiation. In contrast, the culture of these embryos for 72 h results in subtle abnormalities of the head and the first branchial arch. The second limitation is that the gross morphology and histology are often not informative enough to distinguish between overall toxicity and developmental toxicity. We suggest some improvements by the association of WEC with two specific techniques: 1) whole-mount immunostaining of sensory ganglia and nerves and 2) in situ hybridization on histological sections using molecular probes for some developmental genes. Embryos reaching about the 30 somite stage at the end of the culture were processed for whole-mount immunostaining of sensory ganglia and nerves. We show that these structures are very sensitive to the noxious effects of HgCl2 and valproate. Both developmental retardations and dysmorphogeneses of the cervical ganglia and nerves were observed. Embryos were also exposed in vitro to low concentrations of all-trans-retinoic acid (AT-RA) and processed for in situ hybridization with radiolabeled anti-sense RNA probes for the Hoxb-1 and Hoxb-2 developmental genes. Three-dimensional reconstructions of the expression domains were performed. The data show that AT-RA induces ectopic expression domains of Hoxb-1. Our experiments demonstrate that techniques such as immunostaining and in situ hybridization can significantly expand the information obtained from whole postimplantation embryo culture.

Teratogenic effects of selenium compounds on cultured postimplantation rat embryos.

Embryonic susceptibility to selenium (Se) teratogenicity was examined in rats using postimplantation embryo culture. Rat embryos at day 9.5 of gestation were cultured by the roller bottle method for 48 hr in the presence of Se compounds. Sodium selenite, sodium selenate, seleno-DL-methionine, and seleno-DL-cystine were embryolethal at 20, 300, 1,000, and 1,000 microM Se, respectively. All of these compounds caused abnormalities such as deformed optic vesicle and swollen rhombencephalon in the viable embryos. These abnormalities were considered to correspond to in vivo malformations caused by Se in hamster fetuses or in bird embryos. These results indicate that rat embryos are susceptible to Se teratogenicity. It seems that there are differences in potency ranking of Se compounds between rat and bird embryos.

Mouse Whole-embryo Culture in Serum Diluted with Waymouth Medium: A Study of Valproic Acid Teratogenicity

The culture of whole post-implantation rodent embryos has become an important tool in developmental biology and toxicology. Since the establishment of this system, rat serum has been the main culture medium used. In this study, we demonstrate that medium composed of 50% serum and 50% chemically defined medium (Waymouth 705/1) permits satisfactory development of mouse embryos over a 26-hour period. In addition, our data demonstrate that, with less than 50% serum, the frequency of malformations increases and growth and differentiation decrease with dilution in a dose-dependent way. Little information is available in the literature on the types of abnormalities induced by inadequate dilutions of serum. This study shows that a chemically defined medium, supplemented with amounts of serum that are below threshold levels, interferes mainly with the normal development of the head (neural tube, eyes and maxillary processes) and with the growth of the embryo. The second part of this work compares the teratogenicity of valproic acid (VPA) in this new medium to that in undiluted serum. We show that the effects of VPA are similar in terms of the type of alterations observed, the reduction of growth and the differentiation. However, the concentrations necessary to induce these effects in Waymouth/human serum/rat serum are half of those needed in human serum/rat serum.

Whole embryo culture and toxicity testing

The use of post-implantation embryo culture in toxicity testing has been the subject of research as well as debate. Advantages of the method include reduced animal use, and reduced cost and time in comparison with in vivo testing. In addition, the method yields many developmental endpoints: it allows the direct and controlled addition of small amounts of compounds, and the incorporation of metabolizing systems is possible. Disadvantages include the technical skill required, the restricted culture duration, the artificial route of administration of test compounds and the absence of the maternal compartment (and hence the absence of a measure for adult toxicity). Several studies using a variety of compounds have shown promising results with respect to correlations between in vivo effects and the outcome of embryo culture. The question of how a meaningful validation study should be designed is still a matter of dispute. Issues under discussion include: the purpose of validation, culture conditions, endpoint definition, choice of compounds to be tested, the status of in vivo data on test compounds, presentation of results, double-blind and interlaboratory design, and the position of the test within a testing strategy. The validity of whole embryo culture as a toxicity screening test is likely to vary considerably between classes of compounds. Therefore, validation studies with larger sets of related compounds may be more meaningful than those with many unrelated compounds. Whole embryo culture may provide a significant contribution to risk assessment by use in screening, and for mechanistic, structure-activity, and dose-response studies.

Cytochrome P-450-dependent biotransformation of QA 208-199 in cultured rat conceptuses

It has been shown, using the method of rat post-implantation embryo culture, that the rat conceptus metabolizes the lipoxygenase inhibitor N-hydroxy- N-methyl-7-propoxy-2-naphthalenethan-amine (QA 208-199, QAB) in vitro. The capacity to metabolize QAB and accumulate its main metabolites depends on the developmental stage and length of exposure. In the present study, pregnant Han Wistar dams were given ip injections of either Aroclor 1254 (AC), 3-methylcholanthrene (3-MC) or phenobarbital (PB) before the dissection and culture of 9.5- and 10.5-day-old conceptuses in order to identify possible inducible conceptal cytochrome P-450 species. Conceptuses preinduced in utero were exposed to QAB in vitro for 48 hr, after which metabolites in the culture medium and conceptual tissues were determined with HPLC. Embryotoxic effects were evaluated in the same conceptuses. Preinduction in utero with AC, 3-MC or PB revealed that pretreatment with PB led, after 48 hr of culture, to a statistically significantly increased impairment of embryonic differentiation in 9.5-day-old conceptuses in vitro in comparison with vehicle-induced QAB-exposed conceptuses. Preinduction with PB led to higher levels of QAA, the main metabolite in vivo, in the culture medium. In the conceptal tissues, metabolite levels were significantly lower after preinduction with AC. In vehicle-induced 10.5-day-old embryos QAB caused less embryotoxicity in comparison with 9.5-day-old ones. In all preinduced conceptuses cultured from 10.5 to 12.5 days, yolk-sac vascularization was less affected and fewer embryonic anomalies were found in comparison with conceptuses cultured from 9.5 to 11.5 days. AC and PB pretreatment caused a decreased morphological score without changes in overall growth. Analysis of QAB and its metabolites in culture media and conceptal tissues at 12.5 days showed different levels of metabolites depending on the inducer used. To find further evidence for the involvement of cytochrome P-450 enzymes in the conceptal metabolism of QAB, conceptuses preinduced in utero (3-MC or PB) were exposed to QAB in vitro and gassed during the second half of the culture period with a mixture containing 35% carbon monoxide (CO), an inhibitor of cytochrome P-450 enzymes. CO treatment led to an inhibition of conceptal metabolism of QAB in comparison with that in conceptuses cultured under normal gassing conditions (without CO). These results strongly suggest the involvement of cytochrome P-450-dependent monooxygenases in the conceptal metabolism of QAB in vitro. 10.5-day-old conceptuses, after preexposure in utero to PB, were less susceptible to QAB embryotoxicity in vitro than were 9.5-day-old ones. In addition, the response to preinduction appeared to be age dependent.

10] Isolation and culture of whole postimplantation embryos and germ layer derivatives

10] Isolation and culture of whole postimplantation embryos and germ layer derivatives

The culture of postimplantation embryos

Culture methods for postimplantation embryos are now widely used in studies of embryo physiology, growth and development. Available methods support growth and development of rat and mouse embryos at all stages of organogenesis. The best results are obtained from embryos between head fold and early limb bud stages; overall growth and differentiation of these embryos in vitro is almost identical to that in vivo. Some examples of the application of postimplantation embryo culture to specific lines of study are given and some possible future developments of the technique are discussed.

Rodent postimplantation embryo culture: interlaboratory validation as a screening test for teratogenic compounds: Piersma AH,∗ Attenon J,‡ Govers MJAP,∗ van Maele-Fabry G,† Peters PWJ,∗ Picard J,† Schmid BP,‡ Stadler J,§ Verhoef A,∗ Verseil C.§ ∗Natl. Inst. Publ. Health. Env. Prot., Bilthoven, Netherlands; †Cathol. Univ., Louvain-la-Neuve, Belgium; ‡Zyma SA, Nyon, Switzerland; §Pfizer Res. Center, Amboise, France

Rodent postimplantation embryo culture: interlaboratory validation as a screening test for teratogenic compounds: Piersma AH,∗ Attenon J,‡ Govers MJAP,∗ van Maele-Fabry G,† Peters PWJ,∗ Picard J,† Schmid BP,‡ Stadler J,§ Verhoef A,∗ Verseil C.§ ∗Natl. Inst. Publ. Health. Env. Prot., Bilthoven, Netherlands; †Cathol. Univ., Louvain-la-Neuve, Belgium; ‡Zyma SA, Nyon, Switzerland; §Pfizer Res. Center, Amboise, France

Rat post-implantation culture: Production of 5–8-somite embryos by a time-controlled mating procedure

The use of in vitro studies allows a reduction in the number of test animals needed to perform a study. In 24-hr post-implantation embryo culture (from day 10 to day 11 post-insemination) the 5–8 somite stage is considered to be the best starting point. A time-controlled mating procedure has been developed using a positive lordosis reaction and a short period for mating to synchronize pregnancy in rats. With this procedure, 243 hr post-mating 97% of embryos are in a stage of development ranging between 5 and 8 somites. The pregnancy rate (96%) and embryo viability rate (93.4%) are excellent. In these conditions only two or three females are needed to obtain 20 embryos at the required somitic stage. Compared with commonly used mating procedures, this technique reduces considerably the number of females needed for each experiment and workload involved. Furthermore, it allows better planning of whole embryo culture studies and can be easily adapted to obtain embryos at any somitic stage.