Cultured Mouse Embryos Research Articles

Successful Laboratory Transition to a New Single-Stage Time-Lapse Medium (G-TL™) for All Embryo Culture

A newly developed single-stage medium for time-lapse culturing (G-TL™, Vitrolife®) was designed specifically to eliminate a “medium refresh” on Day 3, obviating the need to manipulate/transition embryos into a new culture dish with a second-stage medium. G-TL was extensively evaluated by multiple quality control methods (temperature, pH and 1-cell mouse embryo culture) inside our laboratory prior to developing and initiating the protocol of transitioning all of our embryo culture.

Rho-kinase-dependent actin turnover and actomyosin disassembly are necessary for mouse spinal neural tube closure.

ABSTRACTThe cytoskeleton is widely considered essential for neurulation, yet the mouse spinal neural tube can close despite genetic and non-genetic disruption of the cytoskeleton. To investigate this apparent contradiction, we applied cytoskeletal inhibitors to mouse embryos in culture. Preventing actomyosin cross-linking, F-actin assembly or myosin II contractile activity did not disrupt spinal closure. In contrast, inhibiting Rho kinase (ROCK, for which there are two isoforms ROCK1 and ROCK2) or blocking F-actin disassembly prevented closure, with apical F-actin accumulation and adherens junction disturbance in the neuroepithelium. Cofilin-1-null embryos yielded a similar phenotype, supporting the hypothesis that there is a key role for actin turnover. Co-exposure to Blebbistatin rescued the neurulation defects caused by RhoA inhibition, whereas an inhibitor of myosin light chain kinase, ML-7, had no such effect. We conclude that regulation of RhoA, Rho kinase, LIM kinase and cofilin signalling is necessary for spinal neural tube closure through precise control of neuroepithelial actin turnover and actomyosin disassembly. In contrast, actomyosin assembly and myosin ATPase activity are not limiting for closure.

Behavior and brain gene expression changes in mice exposed to preimplantation and prenatal stress.

Preimplantation culture of mouse embryos has been suggested to result in reduced anxiety-like behavior in adulthood. Here, we investigated the effects of in vitro fertilization (IVF), embryo culture, and different diets on anxiety-like behavior using the elevated plus maze (EPM). We hypothesized that exposure to suboptimal conditions during the preimplantation stage would interact with the suboptimal diet to alter behavior. The expression of genes related to anxiety was then assessed by quantitative real-time polymerase chain reaction in various brain regions. When fed a normal diet during gestation and a moderately high-fat Western diet (WD) postnatally, naturally conceived (NC) and IVF mice showed similar anxiety-like behavior on the EPM. However, when fed a low-protein diet prenatally and a high-fat diet postnatally (LP/HF), NC mice showed a modest increase in anxiety-like behavior, whereas IVF mice showed the opposite: a strongly reduced anxiety-like behavior on the EPM. The robust reduction in anxiety-like behavior in IVF males fed the LP/HF diets was, intriguingly, associated with reduced expression of MAO-A, CRFR2, and GABA markers in the hypothalamus and cortex. These findings are discussed in relation to the developmental origin of health and disease hypothesis and the 2-hit model, which suggests that 2 events, occurring at different times in development, can act synergistically with long-term consequences observed during adulthood.

Maneb-induced dopaminergic neuronal death is not affected by loss of mitochondrial complex I activity: results from primary mesencephalic dopaminergic neurons cultured from individual Ndufs4+/+ and Ndufs4-/- mouse embryos.

Primary cultures from embryonic mouse ventral mesencephalon are widely used for investigating the mechanisms of dopaminergic neuronal death in Parkinson's disease models. Specifically, single mouse or embryo cultures from littermates can be very useful for comparative studies involving transgenic mice when the neuron cultures are to be prepared before genotyping. However, preparing single mouse embryo culture is technically challenging because of the small number of cells present in the mesencephalon of each embryo (150 000-300 000), of which only 0.5-5% are tyrosine hydroxylase-positive, dopaminergic neurons. In this study, we optimized the procedure for preparing primary mesencephalic neuron cultures from individual mouse embryos. Mesencephalic neurons were dissociated delicately, plated on Aclar film coverslips, and incubated in DMEM supplemented with fetal bovine serum for 5 days and then N2 supplement was added for 1 day, which resulted in the best survival of dopaminergic neurons from each embryo. Using this optimized method, we prepared mesencephalic neuron cultures from single Ndufs4 or Ndufs4 embryos and investigated the role of mitochondrial complex I in maneb-induced dopamine neuron death. Our results suggest that maneb toxicity to dopamine neurons is not affected by the loss of mitochondrial complex I activity in Ndufs4 cultures.

Optimal human serum albumin (HSA) concentration in mouse embryo culture systems

Optimal human serum albumin (HSA) concentration in mouse embryo culture systems

Use of a Mouse In Vitro Fertilization Model to Understand the Developmental Origins of Health and Disease Hypothesis

The Developmental Origins of Health and Disease hypothesis holds that alterations to homeostasis during critical periods of development can predispose individuals to adult-onset chronic diseases such as diabetes and metabolic syndrome. It remains controversial whether preimplantation embryo manipulation, clinically used to treat patients with infertility, disturbs homeostasis and affects long-term growth and metabolism. To address this controversy, we have assessed the effects of in vitro fertilization (IVF) on postnatal physiology in mice. We demonstrate that IVF and embryo culture, even under conditions considered optimal for mouse embryo culture, alter postnatal growth trajectory, fat accumulation, and glucose metabolism in adult mice. Unbiased metabolic profiling in serum and microarray analysis of pancreatic islets and insulin sensitive tissues (liver, skeletal muscle, and adipose tissue) revealed broad changes in metabolic homeostasis, characterized by systemic oxidative stress and mitochondrial dysfunction. Adopting a candidate approach, we identify thioredoxin-interacting protein (TXNIP), a key molecule involved in integrating cellular nutritional and oxidative states with metabolic response, as a marker for preimplantation stress and demonstrate tissue-specific epigenetic and transcriptional TXNIP misregulation in selected adult tissues. Importantly, dysregulation of TXNIP expression is associated with enrichment for H4 acetylation at the Txnip promoter that persists from the blastocyst stage through adulthood in adipose tissue. Our data support the vulnerability of preimplantation embryos to environmental disturbance and demonstrate that conception by IVF can reprogram metabolic homeostasis through metabolic, transcriptional, and epigenetic mechanisms with lasting effects for adult growth and fitness. This study has wide clinical relevance and underscores the importance of continued follow-up of IVF-conceived offspring.

Dynamic responses of endothelial cells to changes in blood flow during vascular remodeling of the mouse yolk sac

Despite extensive work showing the importance of blood flow in angiogenesis and vessel remodeling, very little is known about how changes in vessel diameter are orchestrated at the cellular level in response to mechanical forces. To define the cellular changes necessary for remodeling, we performed live confocal imaging of cultured mouse embryos during vessel remodeling. Our data revealed that vessel diameter increase occurs via two distinct processes that are dependent on normal blood flow: vessel fusions and directed endothelial cell migrations. Vessel fusions resulted in a rapid change in vessel diameter and were restricted to regions that experience the highest flow near the vitelline artery and vein. Directed cell migrations induced by blood flow resulted in the recruitment of endothelial cells to larger vessels from smaller capillaries and were observed in larger artery segments as they expanded. The dynamic and specific endothelial cell behaviors captured in this study reveal how sensitive endothelial cells are to changes in blood flow and how such responses drive vascular remodeling.

Distinct adult metabolic consequences following ovarian stimulation versus in vitro culture of mouse embryos

Risk factors for type 2 diabetes may be increased in individuals conceived by in vitro fertilisation (IVF). It is unclear whether these effects are intrinsic to the infertility, or due to controlled ovarian hyperstimulation and/or in vitro culture of the embryo. To delineate the cause, this study metabolically phenotyped adult male mice conceived by natural conception, by ovarian stimulation alone or by IVF.

Effects of β-carotene in cultured mouse embryos exposed to nicotine

The present study investigates the effects of Bisphenol A on the induction of dominant lethal mutation and male reproductive functions. The male rats were gavaged with BPA (10 μg, and 5.0 mg/kg/bw) over a period of six days and control group with vehicle. Each male was cohabited with untreated females sequentially over the period of eight weeks. The mated females were sacrificed on 15th day of gestation. The results revealed a significant increase in dominant lethal mutation rate during fourth and sixth week of mating intervals at 5.0 mg/kgbw dose of BPA. These findings demonstrate that mid-spermatids and spermatocytes are more sensitive to BPA exposure. The male rats sacrificed at the end of mating study showed an increase in the sperm DNA damage, and decrease in motility at higher dose. However, significant reductions in sperm production effects were observed at both lower and higher doses of BPA. These preliminary results indicate that BPA may be a weak male germ cell mutagen.

The effect of vitrification and in vitro culture on the adenosine triphosphate content and mitochondrial distribution of mouse pre-implantation embryos.

The mitochondria are an important source of adenosine triphosphate (ATP) production in pre-implantation embryo. Therefore, the objective of this study was to investigate the effect of vitrification and in vitro culture of mouse embryos on their mitochondrial distribution and ATP content. The embryos at 2-PN, 4-cell and blastocyst stages were collected from the oviduct of stimulated pregnant mice and uterine horns. Then, the embryos were vitrified with the cryotop method using ethylene glycol and dimethylsulphoxide. After evaluating the survival rates of vitrified embryos, their development to hatching stages were assessed. The ATP content of collected in vivo and in vitro embryos at different stages was measured by luciferin-luciferase bioluminescence assay. The distribution of mitochondria was studied using Mito-tracker green staining under a fluorescent microscope. The survival rates of vitrified embryos at 2-PN, 4-cell and early blastocyst stages were 84.3, 87.87 and 89.89%, respectively. The hatching rates in previous developmental stages in vitrified group were 57.44, 66.73 and 70.89% and in non-vitrified group were 66.32, 73.25 and 75.89%, respectively (P>0.05). The ATP content of in vivo or in vitro collected embryos was not significantly different in both vitrified and non-vitrified groups (P>0.05). Mitochondrial distribution of vitrified and non-vitrified 2-PN embryos was similar, but some clampings or large aggregation of mitochondria within the vitrified 4-cell embryos was prominent. Vitrification method did not affect the mouse embryo ATP content. Also, the cellular stress was not induced by this procedure and the safety of vitrification was shown.

Emodin Prevents Ethanol‐Induced Developmental Anomalies in Cultured Mouse Fetus through Multiple Activities

Maternal alcohol ingestion on pregnant period causes fetal alcohol syndrome including psychological and behavioral problems, and developmental abnormality. In this study, we investigated the effect of emodin, an active anthraquinone component found in the roots and bark of the genus Rhamnus (Buckthorn), on ethanol-induced teratogenesis during embryonic organogenesis. We cultured mouse embryos on embryonic day 8.5 for 2 days with ethanol (5 μl/3 ml) and/or emodin (1×10(-5) and 1×10(-4) μg/ml) using a whole embryo culture system and then investigated the developmental evaluation, superoxide dismutase (SOD) activity, and expression patterns of cytoplasmic SOD (SOD1), mitochondrial SOD (SOD2), cytosolic glutathione peroxidase (cGPx), tumor necrosis factor-α (TNF-α), caspase 3, and hypoxia inducible factor 1α (HIF-1α). Morphological parameters, including growth in yolk sac and fetal head, body length, and development of the central nervous system, circulation system, sensory organs, skeletal system, and limbs in embryos exposed to ethanol were significantly decreased compared to those of the normal control group, but co-treatment with emodin (1 × 10(-5) and 1 × 10(-4) μg/ml) significantly improved these parameters. Furthermore, the reduced levels of SOD activity, and SOD1, SOD2, cGPx, and HIF-1α and the increased gene levels of TNF-α and caspase-3 due to ethanol exposure were significantly restored by cotreatment with emodin. Birth Defects Res (Part B) 98:268-275, 2013. © 2013 Wiley Periodicals, Inc. This study revealed that cotreatment with emodin significantly prevented teratogenesis induced by ethanol, not only by modulating hypoxia and antioxidant enzymes, but also by attenuating the enhanced levels of TNF-α and caspase 3 in cultured embryos. Therefore, emodin may be an effective preventive agent for ethanol-induced teratogenesis.

L-Carnitine Supplementation During Vitrification of Mouse Oocytes at the Germinal Vesicle Stage Improves Preimplantation Development Following Maturation and Fertilization In Vitro

Oocyte cryopreservation is important for assisted reproductive technologies (ART). Although cryopreservation of metaphase II (MII) oocytes has been successfully used, MII oocytes are vulnerable to the damage inflicted by the freezing procedure. Cryopreservation of germinal vesicle stage oocytes (GV-oocytes) is an alternative choice; however, blastocyst development from GV-oocytes is limited largely due to the need for in vitro maturation (IVM). Herein, we evaluated the effects of l-carnitine (LC) supplementation during vitrification and thawing of mouse GV-oocytes, IVM, and embryo culture on preimplantation development after in vitro fertilization (IVF). We first compared the rate of embryonic development from the oocytes that had been collected at the GV stage from three mouse strains, (B6.DBA)F1, (B6.C3H)F1, and B6, and processed for IVM and IVF, as well as that from the oocytes matured in vivo, i.e. ovulated (IVO). Our results demonstrated that the rate of blastocyst development was the highest in the (B6.DBA)F1 strain and the lowest in the B6 strain. We then supplemented the IVM medium with 0.6 mg/ml LC. The rate of blastocyst development improved in the B6 but not in the (B6.DBA)F1 strain. Vitrification of GV-oocytes in the basic medium alone reduced the rate of blastocyst development in both of those mouse strains. LC supplementation to the IVM medium alone did not change the percentage of blastocyst development. However, LC supplementation to both vitrification and IVM media significantly improved blastocyst development to the levels comparable with those obtained from vitrified/thawed IVO oocytes in both of the (B6.DBA)F1 and B6 strains. We conclude that LC supplementation during vitrification is particularly efficient in improving the preimplantation development from the GV-oocytes that otherwise have lower developmental competence in culture.

Teratogenicity of Staphylococcus aureus L-forms using a mouse whole-embryo culture model

Our previous studies have suggested that Staphylococcus aureus L-forms are able to pass through the placental barrier of mice from the maternal side to the fetal body and affect fetal growth and development, but little is known about the direct influence of S. aureus L-forms on embryos during the critical period of organogenesis. Mouse embryos at gestational day 8.5 were cultured in vitro for 48 h with 0, 50, 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1). At the end of the culture period, the mouse embryos were assessed morphologically for viability, growth and development. Bacteriological and immunohistochemical staining were used to determine the existence of S. aureus L-forms in embryonic tissues. We found that both crown-rump length and head length of mouse embryos exposed to S. aureus L-forms at a concentration of 50 c.f.u. ml(-1) were reduced. When the mouse embryos were exposed to 100, 200 or 400 c.f.u. S. aureus L-forms ml(-1), the total morphological score, number of somites, dry embryo weight, yolk sac diameter, crown-rump length and head length were significantly lower than those of the control group. With the increased concentration of S. aureus L-forms in the culture medium, there were fewer normally developed embryos and more embryos with abnormalities or retardation in growth. S. aureus L-forms detected by Gram-staining and immunohistochemical detection of antigen were found in the tissues of embryos infected by S. aureus L-forms. These data suggest that S. aureus L-forms exert a direct teratogenic effect on cultured mouse embryos in vitro.

In Vivo Partial Bystander Study in a Mouse Model by Chronic Medium-Dose-Rate γ-Ray Irradiation

The biological activities of molecules secreted into the serum of mice chronically irradiated with γ rays at low or medium dose rate (L/MDR) have not been well studied. In this work, the bioactive molecules found in the serum of chronically irradiated mice (dose rate: 0.0181 Gy/h) were characterized by a cell-based assay (CBA) using microarrays. This technique can predict changes in cytokine levels in serum by measuring gene expression profiles and analyzing molecular signaling pathways. Gene expression in cultured mouse embryo fibroblasts (MEFs) 1 day after addition of serum from nonirradiated or irradiated mice had different profiles. A high level of expression of lipocalin2 (Lcn2) was induced in MEFs upon addition of serum from MDR irradiated mice, and Lcn2 was used as a marker for identifying secreted molecules in serum. Based on microarray analysis of molecular pathways, we predicted that the enhanced gene expression of Lcn2 in MEFs might be caused by interleukin-1 (IL-1) in the serum of the irradiated mice, and that an IL-1α antibody could completely neutralize the enhanced gene expression of Lcn2 in MEFs. The increase in IL-1α levels in the serum from the irradiated mice was confirmed by ELISA experiments. However, an increase in IL-1β could not be detected. These results indicated that IL-1α was released into the serum of mice chronically exposed to a high dose of γ-ray radiation at MDR. We therefore believe that the CBA method using microarrays will be applicable for the screening of bioactive molecules in serum, which will be useful for detecting various diseases and metabolic changes.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture

ObjectiveThis study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos.MethodsA total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05.ResultsThe rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula≤) and 96 hours (blastocyst≤) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst≤) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups.ConclusionThe rate of embryonic development after 96 hours (hatching blastocyst≤) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.

A microfluidic system supports single mouse embryo culture leading to full-term development

The present study demonstrates the feasibility of application of a microfluidic system for in vitro culture of pre-implantation mouse embryos, with subsequent development to full-term upon embryo transfer. Specifically, embryos cultured in groups in nL volume chambers achieve pre-implantation developmental rates up to 95% (4.5 days after fertilization), while birth rates upon transfer in utero are comparable to conventional droplet culture (∼30%). Importantly, while culturing single embryos in conventional microliter droplets hampers full-term development, mouse embryos cultured individually in a confined microfluidic environment achieve normal birth rates (29–33%) with normal morphology. Furthermore, the refreshment of culture media (dynamic culture) during pre-implantation in the microfluidic system does not impair development to term. These results deliver great promise to studies in developmental biology and human assisted reproductive technologies (ART), as nanoliter culture volumes provided by microfluidics will (1) allow online screening of physical and chemical culture parameters and (2) facilitate the acquisition of physiological data at the single embryo level – essential requisites for the determination of optimal embryo culture conditions.

Signaling axis involving Hedgehog, Notch, and Scl promotes the embryonic endothelial-to-hematopoietic transition

During development, the hematopoietic lineage transits through hemogenic endothelium, but the signaling pathways effecting this transition are incompletely characterized. Although the Hedgehog (Hh) pathway is hypothesized to play a role in patterning blood formation, early embryonic lethality of mice lacking Hh signaling precludes such analysis. To determine a role for Hh signaling in patterning of hemogenic endothelium, we assessed the effect of altered Hh signaling in differentiating mouse ES cells, cultured mouse embryos, and developing zebrafish embryos. In differentiating mouse ES cells and mouse yolk sac cultures, addition of Indian Hh ligand increased hematopoietic progenitors, whereas chemical inhibition of Hh signaling reduced hematopoietic progenitors without affecting primitive streak mesoderm formation. In the setting of Hh inhibition, induction of either Notch signaling or overexpression of Stem cell leukemia (Scl)/T-cell acute lymphocytic leukemia protein 1 rescued hemogenic vascular-endothelial cadherin(+) cells and hematopoietic progenitor formation. Together, our results reveal that Scl overexpression is sufficient to rescue the developmental defects caused by blocking the Hh and Notch pathways, and inform our understanding of the embryonic endothelial-to-hematopoietic transition.

Pharmacological manipulation of blood and lymphatic vascularization in ex vivo–cultured mouse embryos

Formation of new blood and lymphatic vessels is involved in many physiological and pathological processes, including organ and tumor growth, cancer cell metastasis, fluid drainage and lymphedema. Therefore, the ability to manipulate vascularization in a mammalian system is of particular interest to researchers. Here we describe a method for pharmacological manipulation of de novo and sprouting blood and lymphatic vascular development in ex vivo-cultured mouse embryos. The described protocol can also be used to evaluate the properties of pharmacological agents in growing mammalian tissues and to manipulate other developmental processes. The whole procedure, from embryo isolation to image quantification, takes 3-5 d, depending on the analysis and age of the embryos.

The redox state of recombinant human serum albumin and its optimal concentration for mouse embryo culture

Albumin has multiple physiological functions in embryo culture, such as a chelator of heavy metals, free radical scavenger, pH and osmotic regulator, a stabilizer, growth factor carrier, a surfactant, and a nutrient. However, the commercially available human serum albumin (HSA) products may not be completely safe since they could be contaminated with viruses and prions. Recombinant human serum albumin (rHSA) has been reported to be as efficient as commercial HSA for fertilization and embryo development. Despite the possible benefits of rHSA, it has not been widely used for embryo culture due to its high cost of production. Our objective was to analyze the redox state of different types of HSA products and rHSA to define oxidative status batch variations of HSA and rHSA and to evaluate the optimal concentration of rHSA for mouse embryo culture. The redox state of the HSA and rHSA used in embryo culture media was found to vary widely. Redox variations were found among different HSA batches as well as among rHSA batches. The highest blastocyst development and hatching rates were obtained with rHSA used at a concentration of 0.05 mg/mL. We showed that very low concentrations of rHSA were most favorable for advanced mouse embryo development in culture.

Offspring from Mouse Embryos Developed Using a Simple Incubator-Free Culture System with a Deoxidizing Agent

To culture preimplantation embryos in vitro, water-jacketed CO2 incubators are used widely for maintaining an optimal culture environment in terms of gas phase, temperature and humidity. We investigated the possibility of mouse embryo culture in a plastic bag kept at 37°C. Zygotes derived from in vitro fertilization or collected from naturally mated B6D2F1 female mice were put in a drop of medium on a plastic culture dish and then placed in a commercially available plastic bag. When these were placed in an oven under air at 37°C for 96 h, the rate of blastocyst development and the cell numbers of embryos decreased. However, when the concentration of O2 was reduced to 5% using a deoxidizing agent and a small oxygen meter, most zygotes developed into blastocysts. These blastocysts were judged normal according to their cell number, Oct3/4 and Cdx2 gene expression levels, the apoptosis rate and the potential for full-term development after embryo transfer to pseudopregnant recipients. Furthermore, using this system, normal offspring were obtained simply by keeping the bag on a warming plate. This culture method was applied successfully to both hybrid and inbred strains. In addition, because the developing embryos could be observed through the transparent wall of the bag, it was possible to capture time-lapse images of live embryos until the blastocyst stage without needing an expensive microscope-based incubation chamber. These results suggest that mouse zygotes are more resilient to their environment than generally believed. This method might prove useful in economical culture systems or for the international shipment of embryos.