Embryo Culture Studies Research Articles

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium.

BackgroundWhole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis‐stage rodent embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of “replacement, reduction, and refinement” of animals in research (the 3Rs).MethodsWe performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum‐free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure.ResultsA literature review revealed use of both serum‐free and diluted rat serum‐based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum‐free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes.ConclusionRat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.

Expression of TGFβ superfamily components and other markers of oocyte quality in oocytes selected by brilliant cresyl blue staining: relevance to early embryonic development.

Brilliant cresyl blue (BCB) is a super-vital stain that has been used to select competent oocytes in different species. One objective of the present study was to assess the relationship between BCB staining, which correlates with an oocyte's developmental potential, and the transcript abundance for select TGFβ-superfamily components, SMAD2/3 and SMAD1/5 phosphorylation levels, and oocyte (JY1) and cumulus-cell (CTSB, CTSK, CTSS, and CTSZ) transcript markers in bovine oocytes and/or adjacent cumulus cells. The capacity of exogenous follistatin or JY1 supplementation or cathepsin inhibitor treatment to enhance development of embryos derived from low-quality oocytes, based on BCB staining, was also determined. Cumulus-oocyte complexes (COCs) from abattoir-derived ovaries were subjected to BCB staining, and germinal-vesicle-stage oocytes and cumulus cells were harvested from control, BCB+, and BCB- (low-quality oocyte) groups for real-time PCR or Western-blot analysis. Remaining COCs underwent in vitro maturation, in vitro fertilization, and embryo culture in the presence or absence of the above exogenous supplements. Levels of FST, JY1, BMP15, and SMAD1, 2, 3, and 5 transcripts were higher in BCB+ oocytes whereas CTSB, CTSK, CTSS, and CTSZ mRNA abundance was higher in cumulus cells surrounding BCB- oocytes. Western-blot analysis revealed higher SMAD1/5 and SMAD2/3 phosphorylation in BCB+ than BCB- oocytes. Embryo-culture studies demonstrated that follistatin and cathepsin inhibitor treatment, but not JY-1 treatment, improve the developmental competence of BCB- oocytes. These results contribute to a better understanding of molecular indices of oocyte competence.

Embryo culture and embryo rescue studies in wild Musa spp. (Musa ornata)

Embryo culture and embryo rescue studies in wild Musa spp. (Musa ornata)

Therapeutic Drugs that Slow the Heart Rate of Early Rat Embryos. Is there a Risk for the Human?

During the organogenic period of development the cardiovascular system of the embryo fulfills several functions including delivery of oxygen and nutrients and a hemodynamic role necessary for cardiac morphogenesis, angiogenesis and hematopoiesis. It is expected that at each stage of embryonic development there is an ideal embryonic heart rate and contractility that maintains the optimal blood flow and pressure to fulfill these various functions. In vitro rat embryo culture studies have revealed that many therapeutic drugs (antiarrhythmics, antidepressants, antipsychotics and anticonvulsants), that may be taken during human pregnancy, cause a concentrationdependent slowing of the embryonic heart and irregular heart rate at higher concentrations. The concentrations causing bradycardia in vitro are often close to human therapeutic plasma concentrations and raise concern that these drugs can potentially cause embryonic death or malformations, and that current reproductive toxicity testing does not adequately examine possible effects of drugs on the embryo's cardiac function.

Methanol teratogenicity in mutant mice with deficient catalase activity and transgenic mice expressing human catalase

The role of catalase in methanol (MeOH) teratogenesis is unclear. In rodents it both detoxifies reactive oxygen species (ROS) and metabolizes MeOH and its formic acid (FA) metabolite. We treated pregnant mice expressing either high (hCat) or low catalase activity (aCat), or their wild-type (WT) controls, with either MeOH (4g/kg ip) or saline. hCat mice and WTs were similarly susceptible to MeOH-initiated ophthalmic abnormalities and cleft palates. aCat and WT mice appeared resistant, precluding assessment of the developmental impact of catalase deficiency. Catalase activity was respectively increased at least 1.5-fold, and decreased by at least 35%, in hCat and aCat embryos and maternal livers. MeOH and FA pharmacokinetic profiles were similar among hCat, aCat and WT strains. Although the hCat results imply no ROS involvement, embryo culture studies suggest this may be confounded by maternal factors and/or a requirement for higher catalase activity in the hCat mice.

Species‐specificity of ethylene glycol‐induced developmental toxicity: toxicokinetic and whole embryo culture studies in the rabbit

High-dose gavage exposure to ethylene glycol (EG) is teratogenic in rats, but not rabbits. To investigate the reason for this species difference, toxicokinetic and whole embryo culture (WEC) studies were conducted in gestation day 9 New Zealand White rabbits, and the data compared to very similar data previously generated in pregnant rats. In the toxicokinetic study, maximal levels of unchanged EG in rabbits were comparable to those reported for rats. However, maximal levels of EG's teratogenic metabolite, glycolic acid (GA), in rabbit maternal blood and embryo were only 46% and 10% of the respective levels in rats. The toxicokinetic profile suggested that the lower GA levels in rabbits were due to a slower rate of maternal metabolism of EG to GA, slow uptake of GA into the yolk sac cavity fluid which surrounds the embryo, and negligible transfer via the visceral yolk sac (VYS) placenta. In the WEC study, exposure of rabbit conceptuses to high concentrations (< or = 12.5 mM) of GA was without effect, which contrasts with reported effects in rat WEC at > or = 3 mM. Overall, these data implicate toxicokinetics as an important factor underlying the species difference, although intrinsic insensitivity of the rabbit embryo might also be involved. Integration of these findings with published human data suggest that the rabbit is the more relevant model for human EG exposure, based on the negligible role of the rabbit VYS in placental transfer (humans lack a VYS) and similar rates of EG metabolism and extraembryonic fluid turnover.

Embryo culture and long-term consequences

The development ofpre-elongation (ruminants) and preimplantation (e.g. mouse and humans) embryos ex vivo has evolved over the past four decades into a reliable technology that is used as a research tool in developmental biology, as well as other embryo technologies, for application in infertility treatment, species conservation and selective breeding. It is clear from a variety of embryo culture studies that adaptive responses by embryos during culture can lead to significant alterations in subsequent developmental profiles, the mechanisms of which are not entirely clear but are unlikely to be limited to a single mechanism because this does not account for the variability seen in responses and the emerging list of specific cellular stressors that cause long-term deviations in fetal development. Epigenetic mechanisms, especially deviation of methylation patterns, and adaptation via causal pathways linking gene expression signalling with critical developmental time points, especially of placental development, are two candidates. Observational studies on post-transfer consequences must now be designed so that specific candidate pathways are followed to elucidate their role in perturbed development following transfer.

Plant cell and biotechnology studies in Linum usitatissimum – a review

The species Linum usitatissimum (flax/linseed) has been the focus of a great deal of both basic and applied research effort in plant cell and biotechnology studies in recent years. In this review we consider applications of the techniques of plant biotechnology in this species under several distinct headings. Plant cell and tissue regeneration strategies and applications are discussed, and the applications of the techniques of somatic embryogenesis, protoplast isolation, culture and fusion and cell suspension cultures in this species are described. A major area of study is the use of anther and microspore culture where clear advantages to breeding programmes could be applied. In addition, embryo and ovary culture studies have resulted in significant findings. The more recent technologies of gene transfer and expression by genetic transformation are reviewed, and a section on strategies for improvements in technological quality is also included. Finally we propose conclusions and future prospects for this ancient, but still highly relevant crop.

Genetic polymorphisms: Impact on the risk of fetal alcohol spectrum disorders

Clinical reports on monozygotic and dizygotic twins provided the initial evidence for the involvement of genetic factors in risk vulnerability for fetal alcohol spectrum disorders (FASD) including fetal alcohol syndrome (FAS). Research with selectively bred and inbred rodents, genetic crosses of these lines and strains, and embryo culture studies have further clarified the role of both maternal and fetal genetics in the development of FASD. Research to identify specific polymorphisms contributing to FASD is still at an early stage. To date, polymorphisms of only one of the genes for the alcohol dehydrogenase enzyme family, the ADH1B, have been demonstrated to contribute to FASD vulnerability. In comparison with ADH1B*1, both maternal and fetal ADH1B*2 have been shown to reduce risk for FAS in a mixed ancestry South African population. ADH1B*3 appears to afford protection for FASD outcomes in African-American populations. Other candidate genes should be examined with respect to FASD risk, including those for the enzymes of serotonin metabolism, in particular the serotonin transporter. By its very nature, alcohol teratogenesis is the expression of the interaction of genes with environment. The study of genetic factors in FASD falls within the new field of ecogenetics. Understanding of the array of genetic factors in FASD will be enhanced by future genetic investigations, including case-control, family association, and linkage studies.

Glutathione depletion modulates methanol, formaldehyde and formate toxicity in cultured rat conceptuses.

The proposed use of methanol (H3COH) as an alternative to fossil fuels has prompted concern about potential health risks resulting from widespread environmental exposure. Methanol is teratogenic in rodents and, although the exact toxic species is not known, teratogenesis may result from the enzymatic biotransformation of H3COH to formaldehyde (CH2O) and formic acid causing increased biological reactivity and toxicity. A protective role for the antioxidant glutathione (GSH) has been described for H3COH, CH2O and formic acid toxicity in various biological systems but has yet to be evaluated in the developing conceptus. Whole embryo culture studies were conducted using GD 10-11 rat conceptuses to elucidate the relationship between H3COH and its metabolites and GSH status. Methanol exposure produced a decrease in normal growth parameters and a dose-dependent loss of viability. CH2O had deleterious effects on embryo growth and viability. Sodium formate (HCOONa) exposure resulted in a high mortality rate but viable embryos did not manifest any abnormalities. Methanol, CH2O, and HCOONa all produced a significant depletion of GSH in both embryo and VYS. Inhibition of GSH synthesis by L-buthionine-S,R-sulfoximine (BSO) treatment exacerbated H3COH, CH2O and HCOONa embryotoxicity. Interestingly, only H3COH/BSO and CH2O/BSO co-treatments caused increased malformation, while embryos treated with HCOONa/BSO did not produce any developmental deformities. These results implicate CH2O as the most embryotoxic H3COH metabolite, on a molar basis, in terms of causing dysmorphogenesis, alterations of normal growth parameters and embryolethality. HCOONa was selectively embryolethal and did not produce dysmorphogenesis. CH2O toxicity is potentiated by GSH depletion, indicating that GSH may be more directly involved in its detoxication in the embryo.

Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture

Background: The traditional anesthetic used for collection of the serum culture medium for whole rat embryo culture studies has been ether. However ethical concerns have been raised due to the irritant nature of the vapour and safety concerns due to the risk of fire. Methods: Growth and development of gestation day 9.5 rat embryos cultured for 48 h in serum collected from rats anesthetised with either ether, isoflurane or halothane were compared. Results: There were no differences in any of the parameters used to assess embryonic development when embryos were grown in serum collected using either ether or isoflurane anesthetics. However, when embryos grown in serum collected using ether or halothane were compared, embryonic development was similar in all respects, except for a reduced number of embryos turned to become fully dorsally convex in the halothane group ( p<0.05). Conclusions: The data indicate that isoflurane is an appropriate alternative to ether for collection of the serum culture medium for whole rat embryo culture, while halothane may cause some delay of embryonic development.

Efeito de sistema de cultivo, célula somática e soro em co-cultura sobre o desenvolvimento de embriões bovinos fecundados in vitro

O objetivo deste experimento foi o de avaliar o efeito de sistemas de cultivo e de diferentes células somáticas e soro bovino na co-cultura sobre a produção de embriões bovinos fecundados in vitro. No experimento um avaliou-se o efeito do sistema de co-cultura com células da tuba uterina e do sistema "definido". No experimento dois utilizaram-se células da granulosa ou da tuba uterina para a co-cultura em meio CR1aa (Charles Rosenkrans). No experimento três utilizou-se soro de vaca em cio (SVC) ou soro fetal bovino (SFB), ambos em co-cultura com células da granulosa em CR1aa. Os ovócitos utilizados foram obtidos de ovários colhidos em matadouro e maturados in vitro em meio 199 com soro de vaca em cio e FSH por 24h. Após a maturação, os ovócitos foram fecundados in vitro por 22h e posteriormente divididos aleatoriamente nos tratamentos. Avaliaram-se a taxa de clivagem no dia três do cultivo, a produção de blastocistos nos dias sete e oito, e de blastocistos eclodidos nos dias nove e dez. Não houve diferença entre os sistemas em co-cultura e "definido" quanto à taxa de clivagem (80,7% e 75,4%) e de produção de blastocisto (19,4% e 17,7%). Entretanto, a taxa de blastocistos eclodidos foi superior para o sistema em co-cultura (37,5%) quando comparado com o sistema "definido" (8,7%). O cultivo embrionário em células da tuba uterina ou da granulosa resultaram em taxas de clivagem, produção de blastocisto e blastocistos eclodidos semelhantes entre si (65,5% e 66,7% de clivagem, 11,6% e 13,7% de blastocistos e 23,1% e 50,0% de blastocistos eclodidos; P&gt;0,05), bem como o cultivo com SFB ou SVC (63,9% e 70,2% de clivagem, 14,3% e 8,7% de blastocisto e 41,2% e 33,3% de blastocistos eclodidos; P&gt;0,05). Conclui-se que o sistema de cultivo "definido" pode ser utilizado para estudos com cultivo de embriões in vitro, no entanto, os resultados quanto à taxa de eclosão ainda são inferiores ao sistema em co-cultura. As células da granulosa e da tuba uterina possuem efeito semelhante sobre o desenvolvimento embrionário, assim como o soro de vaca em cio e o soro fetal bovino, quando em co-cultura.

Large offspring syndrome and other consequences of ruminant embryo culture in vitro: Relevance to blastocyst culture in human ART

In vitro production of embryos from domestic animals is used to augment conventional genetic improvement programmes in agriculture and to facilitate advances in gene transfer and cloning. However, production of embryos in vitro exposes them to hazards not normally encountered in vivo and, as a result, there have been unforeseen consequences including the large offspring syndrome. This syndrome is manifest as abnormal growth and development at fetal, neonatal and later stages after transfer of embryos cultured in vitro for up to 1 week after fertilization. Our embryo culture and fetal development studies have begun to characterize many of the genetic, metabolic and developmental features associated with the syndrome. This review considers the findings of these studies in the context of blastocyst production in vitro, emphasizing the impact of culture strategies on ruminant (cattle and sheep) embryo composition and developmental competence. The need to alter in vitro production strategies to safeguard oocytes and embryos during culture is discussed. Finally, the implications of experiences gained in domestic animal studies are considered in the context of current options for human embryo culture. The need for an appreciation of the sensitivity of the embryo to its environment and the possible short- and long-term consequences of inappropriate in vitro production strategies are considered.

Specification of left-right asymmetry in mammals: Embryo culture studies of stage of determination and relationships with morphogenesis and growth

The internal mammalian body plan is laterally asymmetric with a consistent handedness such that some organs are placed on one side (stomach on the left, for example) and paired organs are not symmetric (for example, there are more lung lobes on the right). Some chemical teratogens can affect the development of asymmetry, and some can cause asymmetric defects in overtly symmetric structures, but the mechanisms are unknown. We have used chemical treatment of rat embryos in culture to examine the stage at which the left-right axis is determined and show that all effective treatments can affect left-right axis development up to the early headfold stage, but not from late headfold onwards. This suggests that the left-right axis is determined by the late headfold stage, even though the embryo is overtly symmetric at this stage. It appears to be much easier to induce an abnormal left-right axis from late allantoic bud and early headfold stages; than the early allantoic bud stage, but we have not established the earliest stage at which a response can be induced. Complete situs inversus was the most common chemically induced abnormality, although heart looping and body turning could be inverted separately, suggesting that the two phenomena are linked but not wholly interdependent. The treatments appeared to cause a loss of handedness, rather than inducing inversion, since the incidence of an abnormal left-right axis never exceeded 50%. All treatments except methoxamine, an α 1 adrenergic agonist, induced an abnormal left-right axis in association with other morphologic defects and growth retardation. However, there was no relationship between the severity or incidence of dysmorphology, nor growth retardation, and left-right abnormality, suggesting that: although the process that specifies lateral asymmetry is labile, it is independent of general growth and morphogenesis.

Embryonic mechanisms underlying the prevention of neural tube defects by vitamins

Folic acid prevents up to 70% of human neural tube defects (NTD), offering the possibility of effective primary prevention for these crippling birth defects. Recent studies with mouse models of NTD aimed to shed light on two unsolved problems of NTD prevention: 1) the mechanism of action of folic acid, and 2) the development of an alternative therapy to prevent NTD that are unresponsive to folic acid. Using a biochemical screening test in cultured mouse embryos, we identified a defect of folate metabolism in embryos homozygous for the splotch (Sp2H) mutation that develop anencephaly and spina bifida. In contrast, there are no demonstrable abnormalities of folate metabolism in two other mouse mutants, curly tail and loop-tail, which also develop NTD. Exogenous folic acid prevents up to 50% of NTD in homozygous splotch embryos. Thymidine is also effective in preventing a proportion of NTD in this mutant, suggesting an inborn error in the provision of folate intermediates for pyrimidine biosynthesis in splotch mice. NTD in mutant curly tail mice do not respond to folic acid but are preventable in up to 70% of cases by exogenous myo-inositol. Embryo culture studies show this effect to be mediated via stimulation of protein kinase C and upregulation of retinoic acid receptor β expression in the embryonic hindgut. Inositol therapy may represent a possible means of preventing folate-resistant NTD in humans. MRDD Research Reviews 1998;4:264–268. © 1998 Wiley-Liss, Inc.

Embryonic mechanisms underlying the prevention of neural tube defects by vitamins

Folic acid prevents up to 70% of human neural tube defects (NTD), offering the possibility of effective primary prevention for these crippling birth defects. Recent studies with mouse models of NTD aimed to shed light on two unsolved problems of NTD prevention: 1) the mechanism of action of folic acid, and 2) the development of an alternative therapy to prevent NTD that are unresponsive to folic acid. Using a biochemical screening test in cultured mouse embryos, we identified a defect of folate metabolism in embryos homozygous for the splotch (Sp2H) mutation that develop anencephaly and spina bifida. In contrast, there are no demonstrable abnormalities of folate metabolism in two other mouse mutants, curly tail and loop-tail, which also develop NTD. Exogenous folic acid prevents up to 50% of NTD in homozygous splotch embryos. Thymidine is also effective in preventing a proportion of NTD in this mutant, suggesting an inborn error in the provision of folate intermediates for pyrimidine biosynthesis in splotch mice. NTD in mutant curly tail mice do not respond to folic acid but are preventable in up to 70% of cases by exogenous myo-inositol. Embryo culture studies show this effect to be mediated via stimulation of protein kinase C and upregulation of retinoic acid receptor β expression in the embryonic hindgut. Inositol therapy may represent a possible means of preventing folate-resistant NTD in humans. MRDD Research Reviews 1998;4:264–268. © 1998 Wiley-Liss, Inc.

Mechanisms of normal and abnormal neurulation: evidence from embryo culture studies.

The method of whole embryo culture has been used extensively in analyzing the mechanisms underlying formation of the mammalian neural tube. These studies have provided insight into the cell lineage of the various tissues that comprise the neurulation stage embryo, the role of microfilaments, extracellular matrix and cell proliferation in the morphogenetic events of neural tube closure and the action of specific genes and gene products in establishment of the nervous system. This information is of considerable importance not only as a means of elucidating the processes of normal embryogenesis but also to shed light on the pathogenesis of important human birth defects.

Gene expression domains as markers in developmental toxicity studies using mammalian embryo culture.

We are examining the hypothesis that expression domains of developmental control genes may be informative markers in mammalian embryo culture studies of developmental toxicity. Expression domains might be altered directly by chemical exposure, or might reflect developmental abnormality prior to any overt morphological defect. Whole-mount in situ hybridization using digoxygenin-labeled RNA probes was used to monitor the regions of expression of Hoxb-4, Pax-3 and Emx-2. These genes were selected because of their different restrictions within the developing CNS; Hoxb-4 for its anterior margin in the hindbrain, Pax-3 for its dorso-ventral pattern in the spinal cord, and Emx-2 for being restricted to a portion of the forebrain. Valproic acid was used as a prototype developmental toxicant because of its known actions on neural tube closure and on segmentation. For patterns of expression, we made three comparisons, between: rat in vivo developed embryos and published descriptions for mouse; rat cultured and in vivo; control and valproate exposed. For these genes, there were no differences between domains of expression in rat and mouse, nor between rat cultured and in vivo embryos. In valproate-exposed embryos, some domains were spatially abnormal, for example Pax-3 in the neural crest, but this was coincident with structural defects induced by the treatment. There was no indication, for these three genes, and this teratogen, that treatment caused any shifts in boundaries of expression, nor induced any ectopic domains, even though exposures induced overt malformation.

Phenytoin covalent binding and embryopathy in mouse embryos co-cultured with maternal hepatocytes from mouse, rat, and rabbit

The anticonvulsant drug phenytoin is teratogenic in a variety of species including humans. Traditional embryo culture studies have employed the addition of 9000 g supernatant (S-9) or microsomal fractions from induced rat or mouse liver as an exogenous bioactivating system to approximate a maternal contribution. However, cellular fractions, unlike cultured intact hepatocytes, may themselves be embryotoxic, and do not reflect the in vivo balance of bioactivation and detoxification. To evaluate in vitro the known in in vivo differential species susceptibility to phenytoin teratogenesis, day 9.5 (day of plug = day 1) mouse embryos either were cultured alone for 24 hr or were co-cultured with hepatocytes from maternal mice, rats or male rabbits, thereby exposing the embryos to the effects of potential species-specific phenytoin metabolism. In the absence of hepatocytes, phenytoin embryotoxicity was concentration dependent (0, 10, 20 and 60 μg/mL), with decreases in embryonic growth, reflected by reduced yolk sac diameter and crown rump length, apparent within the maternal therapeutic range (20 μg/mL). Covalent binding of the radiolabeled drug to live embryonic tissue was significantly higher than in control embryos previously killed by fixation, suggesting that the embryo can bioactivate phenytoin to a toxic reactive intermediate. Mouse embryos grew equally well with hepatocytes from all three species, indicating interspecies tissue compatibility. The addition of rat and rabbit hepatocytes, but not mouse hepatocytes, significantly enhanced the phenytoin-induced impairment of mouse embryonic development, as demonstrated by reductions in somite number. The phenytoin-induced impairment of mouse embryonic growth was not enhanced by the addition of rat or rabbit hepatocytes, while mouse hepatocytes conferred protection. The covalent binding of phenytoin to extracellular proteins in the culture medium was not enhanced by the addition of mouse hepatocytes. These results suggest that mouse embryos intrinsically can bioactivate phenytoin to a toxic reactive intermediate, with embryopathic consequences. The protection conferred by maternal mouse hepatocytes suggests a species-specific maternal biochemical balance favouring detoxification that is not shared by rat and rabbit hepatocytes, which enhanced phenytoin embryopathy. Thus, while phenytoin teratogenicity likely involves embryonic bioactivation, maternal determinants may contribute variably to teratologic susceptibility in a species-specific manner.

In vitro murine embryotoxicity of cyclophosphamide in embryos co-cultured with maternal hepatocytes: development and application of a murine embryo-hepatocyte co-culture model

The technique of whole embryo culture provides a sensitive model to evaluate both the effects, and their underlying mechanisms, of drugs and environmental chemicals on embryonic development, independent of maternal influences. However, before teratogenic expression, many teratogens must be enzymatically bioactivated to toxic reactive intermediates. To detect such proteratogens, the embryo culture model may need to be coupled with an exogenous bioactivating system if maternal and/or placental metabolism is involved. We developed a similar embryo-hepatocyte co-culture system using embryos and maternal hepatocytes from mice, which often are more sensitive than rats to chemical teratogens, and which may have a balance of phase II drug metabolising enzymes more similar to humans. This murine system was then used to evaluate the relative maternal and embryonic contributions to cyclophosphamide embryopathy. Day 9.5 (morning of plug = day 1) murine embryos were co-cultured for 24 h in vitro with primary cultures of murine maternal hepatocytes (> 85% viability). Murine embryos were exposed to cyclophosphamide concentrations (0, 7.5, 15, 25 μg/ml), similar to those used in rat embryo culture studies. Murine embryos co-cultured with murine maternal hepatocytes developed normally, as did embryos exposed to cyclophosphamide in the absence of hepatocytes. Maternal hepatocytes were necessary for the expression of cyclophosphamide embryotoxicity, which was concentration-dependent, as demonstrated by increasing severity of reductions in crown rump length, yolk sac diameter and somite number. These results show that the co-culture of murine maternal hepatocytes and embryos is feasible, and suggest that maternal bioactivation is required for murine cyclophosphamide embryopathy.