Cultured Mouse Embryos Research Articles

Asymmetric Fate of the Posterior Part of the Second Heart Field Results in Unexpected Left/Right Contributions to Both Poles of the Heart

The second heart field (SHF) contains progenitors of all heart chambers, excluding the left ventricle. The SHF is patterned, and the anterior region is known to be destined to form the outflow tract and right ventricle. The aim of this study was to map the fate of the posterior SHF (pSHF). We examined the contribution of pSHF cells, labeled by lipophilic dye at the 4- to 6-somite stage, to regions of the heart at 20 to 25 somites, using mouse embryo culture. Cells more cranial in the pSHF contribute to the atrioventricular canal (AVC) and atria, whereas those more caudal generate the sinus venosus, but there is intermixing of fate throughout the pSHF. Caudal pSHF contributes symmetrically to the sinus venosus, but the fate of cranial pSHF is left/right asymmetrical. Left pSHF moves to dorsal left atrium and superior AVC, whereas right pSHF contributes to right atrium, ventral left atrium, and inferior AVC. Retrospective clonal analysis shows the relationships between AVC and atria to be clonal and that right and left progenitors diverge before first and second heart lineage separation. Cranial pSHF cells also contribute to the outflow tract: proximal and distal at 4 somites, and distal only at 6 somites. All outflow tract-destined cells are intermingled with those that will contribute to inflow and AVC. These observations show asymmetric fate of the pSHF, resulting in unexpected left/right contributions to both poles of the heart and can be integrated into a model of the morphogenetic movement of cells during cardiac looping.

Culture of mouse embryos on soft environment improves embryo development

Cells are remarkably sensitive to the biophysical cues of their microenvironment. Altering the substrate where cells grow can dramatically change their developmental fate. The standard substrate for embryo culture, the polystyrene dish (PD), has a stiffness of 1 GPa, six order of magnitude greater than the stiffness of epithelia (1KPa), the physiological surface where embryos grow.

Molecular studies of the congenital malformation induced by Largehead Atractylodes Rhizome, the most commonly used Chinese medicine for threatened miscarriage

Largehead Atractylodes Rhizome (LAR) is the most commonly used Chinese herbal medicine for threatened miscarriage. Potential reproductive toxicity of LAR was identified in early pregnancy in animals. Skeletal anomalies including loss of ulna and distal digits, shortening of humerus and radius were observed in higher clinical dose groups. Here, we aimed to study the molecular mechanism of the congenital malformation induced by LAR. In vitro whole mouse embryo culture was used to confirm the embryotoxicity effects of LAR on developing limb buds during early organogenesis. A pregnant mouse model was employed to study the developmental gene expression by quantitative PCR and whole hybridization and apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling staining, in the forelimbs and hindlimbs during development in vivo. Severe growth retardation, multiple embryonic malformations and delayed limb bud development were observed. Limb-specific Tbx gene expressions in both developing forelimbs and hindlimbs were significantly decreased. Increased developmental apoptosis in apical ectodermal ridge and mesenchymal mesoderm of the developing limb buds was identified. Overexpressions of Tbx2 and Tbx3 in embryos in vitro rescued LAR-induced abnormal limb development and reduced apoptosis in the developing forelimb buds. In conclusion, LAR affects limb development by suppressing the expression of limb developmental genes and disturbing programmed cell death during limb formation in mice.

Male Mice Produced by In Vitro Culture Have Reduced Fertility and Transmit Organomegaly and Glucose Intolerance to Their Male Offspring1

It has been reported that suboptimal in vitro culture (IVC) of mouse embryos can affect the postnatal expression of epigenetically sensitive alleles, resulting in altered postnatal growth, organ dimensions, health, and behavior in the offspring. Although these detrimental impacts on the offspring are well described, the relative contribution of the IVC-produced fathers is unclear. In this work, we have analyzed if suboptimal IVC (achieved by altering the culture medium by the addition of FCS) can affect male fertility and if organ size and glucose clearance, two of the adverse effects produced by suboptimal IVC conditions, were transmitted to the next two generations. IVC-produced males had lower sperm concentrations (5.8 × 10(6) spermatozoa in IVC vs. 14.5 × 10(6) spermatozoa in control), and these sperm exhibited decreased overall motility (49.6% vs. 72.8% in control) and progressive motility (22.6% vs. 32.2% in control). Fertility tests demonstrated that the percentage of pregnancies was reduced for IVC males (35% for IVC-produced males vs. 86% for in vivo controls). These features were related to a modified gene expression pattern in adult male testes, showing an altered gene expression in genes involved in DNA repair and apoptosis that was confirmed by TUNEL assay. Regarding the IVC related adverse phenotype transmitted to offspring, male glucose intolerance was shown only in F1 and F2 male but not female offspring. The same occurred with male abnormalities in the organ size of the liver, which were transmitted to F1 and F2 males but not to F1 females; moreover, analysis of the F0, F1, and F2 males revealed greater coefficients of variance in body weight and glucose intolerance than the control group. Finally, we analyzed, through gene silencing, the effect of IVC on the mRNA expression at the blastocyst stage for 11 known gene expression modifiers of epigenetic reprogramming. Suboptimal IVC reduced the expression of Kap1, Sox2, Hdac1, Dnmt1, and Dnmt3a, suggesting a molecular epigenetic role for gene expression modifiers in the origin and transmission of these abnormal phenotypes.

Protective effect of resveratrol against nicotine-induced teratogenesis in cultured mouse embryos

Protective effect of resveratrol against nicotine-induced teratogenesis in cultured mouse embryos

Resveratrol prevents nicotine-induced teratogenesis in cultured mouse embryos

Nicotine, a major toxic component in tobacco smoke, leads to severe embryonic damage during organogenesis in embryos. We investigated whether resveratrol would positively influence nicotine-induced teratogenesis in mouse embryos (embryonic day 8.5) cultured for 48h using a whole embryo culture system. Embryos exposed to nicotine (1mM) revealed significantly severe morphological anomalies, increased levels of caspase-3 mRNA and lipid peroxidation, and decreased levels of cytoplasmic superoxide dismutase (SOD), mitochondrial manganese SOD, cytosolic glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, hypoxia-inducible factor 1α, Bcl-xL, and sirtuin1 (SIRT1) mRNAs and SOD activity compared to those in the normal control group. However, when resveratrol (1×10−8μM or 1×10−7μM) was added concurrently to the embryos exposed to nicotine, all the parameters in above improved conspicuously. These findings indicate that resveratrol has a noted protective effect against nicotine-induced teratogenesis in mouse embryos through its antioxidative and anti-apoptotic effects.

Effect of glucose concentration during in vitro culture of mouse embryos on development to blastocyst, success of embryo transfer, and litter sex ratio

A high-glucose concentration in the reproductive tract during early development may result in aberrant embryo or fetal development, with effects that could have a greater impact on one sex than the other. Here, we determine if a high-glucose concentration impacts embryo development and pregnancy outcomes in a sex-specific manner in the mouse. Zygotes were cultured in potassium simple optimized medium, which typically contains 0.2 mM D-glucose, with and without additional glucose supplementation to a concentration of 28 mM. Zygote cleavage and blastocyst rate did not differ between treatments, but total and trophectoderm cell counts were reduced in blastocysts cultured in a high glucose. No differences between sexes nor inner cell mass cell number were observed within each treatment. Blastocysts developed in both media were transferred to recipients. The percentage of blastocysts resulting in viable pups was significantly reduced when the blastocysts were cultured in 28 mM glucose (74 ± 4%, controls vs. 55.8 ± 7.1%, 28 mM glucose), but conceptus loss affected both sexes equally as litter sex ratio did not differ between treatments (52.7% and 52.2% males for controls and high glucose, respectively). Pup body weight at birth was higher for males than females, but was not affected by earlier culture in high glucose. In conclusion, in vitro culture in medium with a glucose concentration approximating that of diabetic serum reduces total and trophectoderm cell numbers at the blastocyst stage and conceptus development to term, but these detrimental effects are not sex-specific.

In Vitro Culture of Mouse Embryos Reduces Differential Gene Expression Between Inner Cell Mass and Trophectoderm

Differences in gene expression and imprinting have been reported, comparing in vivo versus in vitro generated preimplantation embryos. Furthermore, mouse studies have shown that placenta development is altered following in vitro culture. However, the molecular mechanisms underlying these findings are unknown. We therefore isolated trophectoderm (TE) and inner cell mass (ICM) cells from in vivo and in vitro fertilization (IVF) embryos and evaluated their transcriptome using microarrays. We found that the transcriptomes of in vitro produced ICM and TE cells showed remarkably few differences compared to ICM and TE cells of in vivo generated embryos. In vitro fertilization embryos showed a reduced number of TE cells compared to in vivo embryos. In addition, TE of IVF embryos showed significant downregulation of solute transporter genes and of genes involved in placenta formation (Eomesodermin, Socs3) or implantation (Hbegf). In summary, IVF and embryo culture significantly affects the transcriptome of ICM and TE cells.

Protective effect of [6]-gingerol on the ethanol-induced teratogenesis of cultured mouse embryos

Excessive ethanol consumption during pregnancy causes fetal alcohol syndrome. We investigated the effect of [6]-gingerol on ethanol-induced embryotoxicity using a whole embryo culture system. The morphological changes of embryos and the gene expression patterns of the antioxidant enzymes cytosolic glutathione peroxidase (cGPx), cytoplasmic Cu/Zn superoxide dismutase (SOD1), and Mn-SOD (SOD2), and SOD activity were examined in the cultured mouse embryos exposed to ethanol (5 μL/3 mL) and/or [6]-gingerol (1×10(-8) or 1×10(-7) μg/mL) for 2 days. In ethanol-exposed embryos, the standard morphological score of embryos was significantly decreased compared with those of the control (vehicle) group. However, cotreatment of embryos with [6]-gingerol and ethanol significantly improved all of the developmental parameters except crownrump length and head length, compared with those of the ethanol alone group. The mRNA expression levels of cGPx and SOD2, not SOD1, were decreased consistently, SOD activity were significantly decreased compared with the control group. However, the decreases in mRNA levels of antioxidant enzymes and SOD activity were significantly restored to the control levels by [6]-gingerol supplement. These results indicate that [6]-gingerol has a protective effect against ethanol-induced teratogenicity during mouse embryogenesis.

Analysis of Fertility-Related Soluble Mediators in Human Uterine Fluid Identifies VEGF as a Key Regulator of Embryo Implantation

Embryo implantation requires synchronized dialogue between the receptive endometrium and activated blastocyst via locally produced soluble mediators. During the midsecretory (MS) phase of the menstrual cycle, increased glandular secretion into the uterine lumen contains important mediators that modulate the endometrium and support the conceptus during implantation. This study aimed first to identify the growth factor and cytokine profile of human uterine fluid from fertile women during the midproliferative (MP; nonreceptive) and MS (receptive) phases of the cycle, and from women with unexplained infertility during the MS phase. The second aim was to determine important functions of endometrial secretions for embryo implantation. Analysis of uterine fluid using quantitative Luminex assays revealed the presence of over 30 cytokines and growth factors, of which eight [platelet-derived growth factor-AA, TNF-B, soluble IL-2 receptor-A, Fms-like tyrosine kinase 3 ligand, soluble CD40 ligand, IL-7, interferon-A2, and chemokine (C-X-C motif) ligand 1-3] were previously unknown in human uterine fluid. Comparison of the fertile MP, MS, and infertile MS cohorts revealed vascular endothelial growth factor (VEGF) levels are significantly reduced in uterine fluid during the MS phase in women with unexplained infertility compared with fertile women. Functional studies demonstrated that culturing mouse embryos with either MS-phase uterine fluid from fertile women or recombinant human VEGF significantly enhanced blastocyst outgrowth. Furthermore, treatment of human endometrial epithelial cells with uterine fluid or recombinant human VEGF-A significantly increased endometrial epithelial cell adhesion. Taken together, our data support the concept that endometrial secretions, including VEGF, play important roles during implantation. Identifying the soluble mediators in human uterine fluid and their actions during implantation provides insight into interactions essential for establishing pregnancy, fertility markers, and infertility treatment options.

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development. Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.01 mM EDTA, supported efficient pre- and post-implantation development of mouse zygotes to blastocysts and live pups, respectively. At first, modified PZM (mPZM) was compared with other culture media such as M16, CZB and KSOM-AA for its ability to support development of in vivo mouse zygotes to the blastocyst stage. The proportions of zygotes reaching 2-cell (94–99%) and blastocyst (90–96%) stages in mPZM and other media were not different. However, hatching rates of blastocysts were different (P < 0.05); whereas more than 90% of the blastocysts were hatching in mPZM or KSOM-AA, only 60% of the blastocysts did in M16 or CZB media (P < 0.05). Next we compared post-implantation development of in vitro fertilized zygotes developed to blastocysts in mPZM and KSOM-AA. The proportion of blastocysts developing into live pups was not different between mPZM (49%) and KSOM-AA (44%). Finally, we evaluated whether mPZM could be also used as a fertilization medium. Modified PZM containing 5.56 mM of glucose and 0.4% BSA efficiently supported IVF of mouse gametes. The percent of zygotes cleaving to 2-cell (94–98%) and blastocysts (91–93%) stage was not different from zygotes fertilized in human tubal fluid medium. We concluded that modified pig zygotic medium containing a higher potassium concentration than any other commonly used mouse media supported not only culture of mouse embryos, but also efficient IVF of mouse gametes.

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

BackgroundPreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo.MethodsDetermine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy.ResultsPIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control).ConclusionsPIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.

Resistance of CD-1 and ogg1 DNA Repair–Deficient Mice to Thalidomide and Hydrolysis Product Embryopathies in Embryo Culture

Thalidomide (TD) displays remarkable species specificity, causing birth defects (teratogenesis) in humans and rabbits, but not rats or mice; yet, few determinants of species susceptibility have been identified. Also, certain mouse strains are susceptible to the embryopathic effects of some teratogens in embryo culture despite their resistance in vivo. Herein we show that CD-1 mouse embryos in culture are resistant to limb embryopathies caused by TD and two of its hydrolysis products, 2-phthalimidoglutaramic acid and 2-phthalimidoglutaric acid, although all three compounds cause these embryopathies in rabbit embryo culture. These results show that the resistance of CD-1 mice to TD teratogenesis is inherent to the embryo and is not dependent upon maternal factors, including differential in vivo exposure to the many hydrolysis products of TD. In utero TD exposure of rabbit but not mouse embryos elevates levels of the teratogenic oxidative DNA lesion 8-oxoguanine, which is repaired by oxoguanine glycosylase 1 (OGG1). However, DNA repair-deficient ogg1 knockout mice proved resistant to TD-initiated embryopathies in culture and teratogenesis in vivo, indicating that the resistance of mice is not due to a higher level of DNA repair.

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system(1), allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques(2). The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period(2). Representative results using the tauGFP mouse line are demonstrated.

Development of a Mouse Model for Studying the Effect of Embryo Culture on Embryonic Stem Cell Derivation

For most of the derived human embryonic stem cell (ESC) lines thus far, the majority of human embryos used have been frozen in liquid nitrogen at or prior to the compacting stage for up to 10 years before human ESC derivation. As such they were grown in media that were relatively simple in their formulation compared with those used today. Here we report that culture of mouse embryos in media similar to these produces blastocysts in which both the inner cell mass cell number and the number of ESC progenitor cells (epiblast cells) in the inner cell mass are reduced compared with blastocysts cultured in a purpose-designed sequential (G1/G2) system commonly used today. Embryos cultured in a simple medium were less likely to attach and generate outgrowths. Further, these outgrowths had increased metabolic activity, which has been linked to differentiation, and altered gene expression. Culture of embryos in a simple medium to the compacting stage followed by culture in G2 to the blastocyst stage reduced some of these effects. However, none were improved to the level seen for culture in G1/G2. These results highlight the influence of embryo culture on embryo quality and pluripotency, which is a key factor in determining ESC isolation efficiencies.

Toxicity of monocrotaline on in vitro cultured mouse embryos

To investigate the fetotoxicity of monocrotaline. Mouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of monocrotaline (100, 50, 25, 12.5 mg x L(-1)) were added into the WEC. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment. Obvious fetotoxicity could be observed in various monocrotaline treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indice, especially in opti-otic organ, mandible and bud. Monocrotaline had obvious fetotoxicity in vitro WEC, indicating that exposure of pregnant mice to monocrotaline may have potential risk on fetus.

132 EFFECTS OF OXYGEN TENSION, MEDIUM, AND WELL OF THE WELL ON IN VITRO DEVELOPMENT OF THE MOUSE EMBRYO

The objectives of the present research were to investigate whether embryo culture media have preferences for oxygen tension, to explore the feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate the effect of well of the well (WOW) culture on in vitro preimplantational development of mouse embryos. The results are as follows. First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM regardless of using 3 gas phases: 4% CO2 + 16% O2 + 78% N2 + 2% H2O (lung air), 5% CO2 + 5% O2 + 90% N2 (5% O2, low oxygen), and 5% CO2 + 95% air (20% O2, high oxygen; P &gt; 0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than those in mKSOM when the lung air was used (P &lt; 0.05). Second, when mKSOM was used as the basic medium, the blastocyst rate (22.6%) in the 5% O2 gas phase was notably higher than that in other 2 gas phases (P &lt; 0.05). Third, for the CZB medium, the blastocyst rate was not different significantly among 3 gas environments (P &gt; 0.05). Fourth, both the blastocyst rate (74.6 ± 5.1%) and the mean total cell numbers per blastocyst (76 ± 2) cultured in the WOW system were obviously higher than those in the group culture system (38.2 ± 6.6% and 58 ± 4). Taken together, our results indicate that mKSOM and CZB have preferences for oxygen tension during in vitro culture of mouse embryos, the lung air was reaffirmed to be able to effectively support in vitro preimplantation development of mouse embryos, and the WOW culture system can apparently enhance in vitro developmental competence and blastocyst quality of mouse embryos. L. YS, C. ZB: equal contribution; supported by NSFC (30700574), 863 (2008AA101003).