Post-implantation Embryos Research Articles

Impaired prenatal development and glycemic status in the offspring of rats with experimental streptozotocin-induced diabetes and their correction with afobazole.

Diabetes mellitus was simulated in rats on gestation day 1 by a single intraperitoneal injection of streptozotocin in doses of 40 and 50 mg/kg. Pregnant females showed increased glucose concentrations n the blood and urine, embryonic developmental disorders, such as tongue protrusion, edema, and skin hyperemia with concomitant vascular damage (hemorrhage, hematoma) as well as pre- and post-implantation embryo loss. Afobazole administered orally in doses of 10 and 50 mg/kg to pregnant rats with streptozotocin-induced diabetes significantly decreased prenatal developmental disorders and pre- and post-implantation embryo loss rate. Afobazole in a dose of 50 mg/kg produced maximum protective effect: in rats receiving 40 mg/kg streptozotocin, post-implantation embryo loss decreased by 14.7 times. Afobazole in doses of 10 and 50 mg/kg significantly reduced blood glucose concentration in pregnant rats and normalized glycemia in 90-day-old male offspring.

Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy.

Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants.

Developmental differences in the expression of FGF receptors between human and mouse embryos

IntroductionFibroblast growth factor (FGF) signaling is essential for early trophoblast expansion and maintenance in the mouse, but is not required for trophectoderm specification during blastocyst formation. This signaling pathway is stably activated to expand the trophoblast stem cell compartment in vivo, while in vitro, FGFs are used for the derivation of trophoblast stem (TS) cells from blastocysts and early post-implantation mouse embryos. However, the function of FGFs during human trophoblast development is not known. MethodsWe sought to derive TS cells from human blastocysts in a number of culture conditions, including in the presence of FGFs and stem cell factor (SCF). We also investigated the expression of FGF receptors (FGFRs) in blastocysts, and the expression of FGFR2 and activated ERK1/2 in first trimester human placentae. ResultsWe found that SCF, but not FGF2/4, improved the quality of blastocyst outgrowths, but we were unable to establish stable human TS cell lines. We observed CDX2 expression in the trophectoderm of fully blastocysts, but rarely observed transcription of FGFRs. FGFR2 protein was not detected in human blastocysts, but was strongly expressed in mouse blastocysts. However, we found robust FGFR2 expression and activated ERK1/2 in the cytotrophoblast layer of early human placenta. DiscussionOur data suggests that initiation of FGF-dependent trophoblast expansion may occur later in human development, and is unlikely to drive maintenance of a TS cell compartment during the peri-implantation period. These findings suggest that cytotrophoblast preparations from early placentae may be a potential source of FGF-dependent human TS cells.

The expression and localization of Crb3 in developmental stages of the mice embryos and in different organs of 1-week-old female mice.

Crumbs homolog 3 (Crb3) is a member of the Crumbs family of proteins. This protein may play a role in epithelial cell polarity and is associated with tight junctions at the apical surface of epithelial cells. Alternative transcriptional splice variants that encode different Crb3 isoforms have been characterized. The expression of Crb3 mRNA and protein was observed in the pre-implantation mouse embryos and different organs of 1-week-old mouse, and Crb3 expression was primarily observed in the cytoplasm. Crb3 was expressed in a unique temporal pattern in pre-implantation embryos. The main characteristic of Crb3 expression was that the positive signals were stronger in the mature oocytes and zygotes than in the 2-cell, 4-cell, and 8-cell stages and the morula, but a similar level of high expression was observed in blastocysts. Therefore, the Crb3 expression signal during the course of development process grew gradually stronger from the 2-cell stage to blastocyst. In addition, Crb3 protein was widely distributed in each stage of the post-implantation embryos. Crb3 expression was observed in the inner cell mass, trophoblast cells and endoderm of E4.5d embryos; in the chorion, amnion, trophoblast cells, yolk sac endoderm and embryo ectoderm of E7.5d embryos; in the amnion and limb bud of E8.0d embryos; and in the semicircular canal epithelium, retina, lens vesicle and liver tissue of E13.5d embryos. Crb3 was expressed at different levels in different organs of 1-week-old mouse with the strengths in the following order: kidney > small intestine > stomach > uterus > liver > skeletal muscle > cerebellum > brain. The presence of Crb3 in many organs and the regularity of Crb3 distribution in the process of mouse embryonic development indicate that Crb3 protein plays an important role in establishing and maintaining the polarity of mouse embryos.

(Invited) Finite Element Model Simulations to Assist the Design of Microdevices Dedicated to the Localized Electroporation of Mouse Embryos

We have recently developed a microsystem to electroporate a few cells at the surface of early post-implantation mouse embryos. We could achieve the efficient, reproducible, and safe transfection of various genetic markers, which allowed single cell fate studies during morphogenesis. However, our single-use polymeric device necessitated to be fabricated in a clean room the day before each experiment. Thus, we here introduce an all-glass chip that any biologist can easily recycle in its laboratory. Most importantly, during the technological evolution process we could validate a comprehensive design strategy based on finite element model simulations. Indeed, both the embryo and the microsystem were represented as very simple electric objects and stationary computations enabled to properly predict the voltage pulse amplitude that would yield optimal device performances.

Invited: Finite Element Model Simulations to Assist the Design of Microdevices Dedicated to the Localized Electroporation of Mouse Embryos

During embryonic development interactions between cell groups enable the growth, migration, and specification of various tissues. The ability to follow and modify cell behavior with accurate spatiotemporal resolution thus appears as a prerequisite to study morphogenesis. More precisely, the dynamics of the above mentioned phenomena can be unraveled by manipulating gene expression locally. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has for instance been harnessed to this aim. However, current strategies suffer from insufficient reproducibility and mediocre survival when applied to early post-implantation mouse embryos. Indeed, between 5.5 and 7.5 days of development theses organisms are small (between 150 and 500 um long) and delicate. Consequently, they require dedicated tools.Sharp metallic electrodes are efficient to concentrate electric field lines and permeate the plasma membrane over a restricted area. Nevertheless, to do so they have to be very close to the targeted tissue. Since harmful species such as gases and protons are produced when voltage pulses are applied, using the preceding microneedles often results in embryo death. Thereby, we introduce here a new type of device to achieve localized electroporation with high efficiency and reduced cell damage. In our “electrodeless” approach the electric field is generated by remote gold pads and channeled towards the cells to be transfected by dielectric tunnels. Finite element model simulations relying on a simple electrical model of the mouse embryo indicated that the present dielectric guide-based design even perform better than the existing alternatives.Such a microsystem was next fabricated on a glass wafer, by patterning the guide walls in SU-8 photoresist, hydrophilizing them with an air plasma, and closing the structure with a Parafilm ceiling. Performances were next tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. More precisely, penetration of a fluorescent dextran enabled to assert membrane permeation, expression of plasmid coding for a fluorescent protein reported on transfection success, and cell death was evaluated thanks to propidium iodide staining. The size of the dielectric guide aperture as well as the voltage pulse sequence could therefore be optimized and transfection could efficiently and reproducibly be restricted to less than four visceral endoderm cells without compromising cell behavior and embryo survival.To demonstrate the validity of our approach with respect to biological questions, we combined targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos to reveal new facts concerning tissue rearrangement between 5.5 and 6.5 days of development. For instance, we could observe that, like the leading DVE cells at the migration front, non-leading ones also send long basal projections and intercalate during their displacement. Thus, they are not passively dragged but they participate actively to the collective motion.Finally, we showed that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation embryos to organ explants dissected from latter stage organisms. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in mouse. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices. Our future work is now dedicated to the fabrication of reusable and robust all-in-glass chip that any embryologist will be able to utilize in his laboratory.

Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms.

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.

Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole.

A microdevice to locally electroporate embryos with high efficiency and reduced cell damage.

The ability to follow and modify cell behaviour with accurate spatiotemporal resolution is a prerequisite to study morphogenesis in developing organisms. Electroporation, the delivery of exogenous molecules into targeted cell populations through electric permeation of the plasma membrane, has been used with this aim in different model systems. However, current localised electroporation strategies suffer from insufficient reproducibility and mediocre survival when applied to small and delicate organisms such as early post-implantation mouse embryos. We introduce here a microdevice to achieve localised electroporation with high efficiency and reduced cell damage. In silico simulations using a simple electrical model of mouse embryos indicated that a dielectric guide-based design would improve on existing alternatives. Such a device was microfabricated and its capacities tested by targeting the distal visceral endoderm (DVE), a migrating cell population essential for anterior-posterior axis establishment. Transfection was efficiently and reproducibly restricted to fewer than four visceral endoderm cells without compromising cell behaviour and embryo survival. Combining targeted mosaic expression of fluorescent markers with live imaging in transgenic embryos revealed that, like leading DVE cells, non-leading ones send long basal projections and intercalate during their migration. Finally, we show that the use of our microsystem can be extended to a variety of embryological contexts, from preimplantation stages to organ explants. Hence, we have experimentally validated an approach delivering a tailor-made tool for the study of morphogenesis in the mouse embryo. Furthermore, we have delineated a comprehensive strategy for the development of ad hoc electroporation devices.

Direct Comparison of the Effects of Slow Freezing and Vitrification on Late Blastocyst Gene Expression, Development, Implantation and Offspring of Rabbit Morulae

This study aimed to assess the effect of different cryopreservation procedures (slow freezing vs vitrification) on the gene expression in pre-implantation embryos and its implication in post-implantation embryo losses in rabbit. For this purpose, rabbit morulae were recovered at Day 3 of development, frozen or vitrified and transferred to recipients. Then, embryos were recovered on Day 3 post-transfer (Day 6 of development) or kept until the end of gestation. Apart from the gene expression analysis at Day 6, we also studied the pre-implantatory and foetal development ability of both cryopreserved embryo types by evaluating late blastocyst development at Day 6, embryo implantation at Day 11 post-transfer (Day 14 of development) and birth rate. We reported that slow freezing and vitrification have similar effects on embryo developmental ability till Day 6, but the distribution of losses changes during implantation and further development. These similarities at Day 6 of development were also reflected in gene expression patterns, and transcriptome analysis showed no differences between frozen and vitrified embryos. Our results confirm that vitrification provides better implantation and birth rates than slow freezing for rabbit embryos. As both the techniques are commonly used in human assisted reproduction, further experiments must be conducted to clarify the causes that may hinder foetal development and their impact on adulthood.

The States of Pluripotency: Pluripotent Lineage Development in the Embryo and in the Dish

The pluripotent cell lineage of the embryo comprises a series of temporally and functionally distinct intermediary cell states, the epiblast precursor cell of the newly formed blastocyst, the epiblast population of the inner cell mass, and the early and late epiblast of the postimplantation embryo, referred to here as early and late primitive ectoderm. Pluripotent cell populations representative of the embryonic populations can be formed in culture. Although multiple pluripotent cell states are now recognised, little is known about the signals and pathways that progress cells from the epiblast precursor cell to the late primitive ectoderm in the embryo or in culture. The characterisation of cell states is most advanced in mouse where conditions for culturing distinct pluripotent cell states are well established and embryonic material is accessible. This review will focus on the pluripotent cell states present during embryonic development in the mouse and what is known of the mechanisms that regulate the progression of the lineage from the epiblast precursor cell and the ground state of pluripotency to the late primitive ectoderm present immediately prior to cell differentiation.

Hazardous effects of sanguinarine on maturation of mouse oocytes, fertilization, and fetal development through apoptotic processes

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre- and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase-3-specific inhibitor effectively blocked sanguinarine-triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase-dependent apoptotic mechanism.

Proteomic analysis of ethanol-induced embryotoxicity in cultured post-implantation rat embryos

Protein expression changes were examined in day 10.5 rat embryos cultured for 24 hr in the presence of ethanol by using two-dimensional electrophoresis and mass spectrometry. Exposure to ethanol resulted in quantitative changes in many embryonic protein spots (16 decreased and 28 increased) at in vitro embryotoxic concentrations (130 and 195 mM); most changes occurred in a concentration-dependent manner. For these protein spots, 17 proteins were identified, including protein disulfide isomerase A3, alpha-fetoprotein, phosphorylated cofilin-1, and serum albumin. From the gene ontology classification and pathway mapping of the identified proteins, it was found that ethanol affected several biological processes involving oxidative stress and retinoid metabolism.

Do all roads lead to Oct4? The emerging concepts of induced pluripotency

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

Oct4/Sox2 Binding Sites Contribute to Maintaining Hypomethylation of the Maternal Igf2/H19 Imprinting Control Region

A central question in genomic imprinting is how parental-specific DNA methylation of imprinting control regions (ICR) is established during gametogenesis and maintained after fertilization. At the imprinted Igf2/H19 locus, CTCF binding maintains the unmethylated state of the maternal ICR after the blastocyst stage. In addition, evidence from Beckwith-Wiedemann patients and cultured mouse cells suggests that two Sox-Oct binding motifs within the Igf2/H19 ICR also participate in maintaining hypomethylation of the maternal allele. We found that the Sox and octamer elements from both Sox-Oct motifs were required to drive hypomethylation of integrated transgenes in mouse embryonic carcinoma cells. Oct4 and Sox2 showed cooperative binding to the Sox-Oct motifs, and both were present at the endogenous ICR. Using a mouse with mutations in the Oct4 binding sites, we found that maternally transmitted mutant ICRs acquired partial methylation in somatic tissues, but there was little effect on imprinted expression of H19 and Igf2. A subset of mature oocytes also showed partial methylation of the mutant ICR, which suggested that the Sox-Oct motifs provide some protection from methylation during oogenesis. The Sox-Oct motifs, however, were not required for erasure of paternal methylation in primordial germ cells, which indicated that the oocyte methylation was acquired post-natally. Maternally inherited mutant ICRs were unmethylated in blastocysts, which suggested that at least a portion of the methylation in somatic tissues occurred after implantation. These findings provide evidence that Sox-Oct motifs contribute to ICR hypomethylation in post-implantation embryos and maturing oocytes and link imprinted DNA methylation with key stem cell/germline transcription factors.

Epigallocatechin gallate induces embryonic toxicity in mouse blastocysts through apoptosis

Catechins, a family of polyphenols found in tea, evoke various responses, including cell death. However, the side effects of these compounds, particularly those on embryonic development, have not been characterized in detail. A previous study by our group showed that (−)-epigallocatechin-3-gallate (EGCG), a catechin highly abundant in green tea, induces different cell-death modes in MCF-7 cells, depending on the treatment dosage. In the current study, we examined the effects of EGCG on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation by embryo transfer. Blastocysts treated with 25–50 μM of EGCG exhibited a significant increase in apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with EGCG was lower than that of their control counterparts. Moreover, in vitro treatment with 25–50 μM of EGCG led to increased resorption of postimplantation embryos and decreased fetal weight. EGCG appeared to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively highlight the potential of EGCG to induce embryonic cytotoxicity.

Mouse Embryonic Stem Cells Established in Physiological-Glucose Media Express the HighKM Glut2 Glucose Transporter Expressed by Normal Embryos

Glut2 is one of the facilitative glucose transporters expressed by preimplantation and early postimplantation embryos. Glut2 is important for survival before embryonic day 10.5. The Glut2 KM (∼16 mmol/liter) is significantly higher than physiologic glucose concentrations (∼5.5 mmol/liter), suggesting that Glut2 normally performs some essential function other than glucose transport. Nevertheless, Glut2 efficiently transports glucose when extracellular glucose concentrations are above the Glut2 KM. Media containing 25 mmol/liter glucose are widely used to establish and propagate embryonic stem cells (ESCs). Glut2-mediated glucose uptake by embryos induces oxidative stress and can cause embryo cell death. Here we tested the hypothesis that low-glucose embryonic stem cells (LG-ESCs) isolated in physiological-glucose (5.5 mmol/liter) media express a functional Glut2 glucose transporter. LG-ESCs were compared with conventional D3 ESCs that had been cultured only in high-glucose media. LG-ESCs expressed Glut2 mRNA and protein at much higher levels than D3 ESCs, and 2-deoxyglucose transport by LG-ESCs, but not D3 ESCs, exhibited high Michaelis-Menten kinetics. Glucose at 25 mmol/liter induced oxidative stress in LG-ESCs and inhibited expression of Pax3, an embryo gene that is inhibited by hyperglycemia, in neuronal precursors derived from LG-ESCs. These effects were not observed in D3 ESCs. These findings demonstrate that ESCs isolated in physiological-glucose media retain a functional Glut2 transporter that is expressed by embryos. These cells are better suited to the study of metabolic regulation characteristic of the early embryo and may be advantageous for therapeutic applications.

Keratin 5-Cre-driven excision of nonmuscle myosin IIA in early embryo trophectoderm leads to placenta defects and embryonic lethality

In studies initially focused on roles of nonmuscle myosin IIA (NMIIA) in the developing mouse epidermis, we have discovered that a previously described cytokeratin 5 (K5)-Cre gene construct is expressed in early embryo development. Mice carrying floxed alleles of the nonmuscle myosin II heavy chain gene (NMHC IIAflox/flox) were crossed with the K5-Cre line. The progeny of newborn pups did not show a Mendelian genotype distribution, suggesting embryonic lethality. Analysis of post-implantation conceptuses from embryonic day (E)9.5 to E13.5 revealed poorly developed embryos and defective placentas, with significantly reduced labyrinth surface area and blood vessel vascularization. These results suggested the novel possibility that the bovine K5 promoter-driven Cre-recombinase was active early in trophoblast-lineage cells that give rise to the placenta. To test this possibility, K5-Cre transgenic mice were crossed with the mT/mG reporter mouse in which activation of GFP expression indicates Cre transgene expression. We observed activation of K5-Cre-driven GFP expression in the ectoplacental cone, in the extraembryonic ectoderm, and in trophoblast giant cells in the E6.5 embryo. In addition, we observed GFP expression at E11.5 to E13.5 in both the labyrinth of the placenta and the yolk sac. NMIIA expression was detected in these same cell types in normal embryos, as well as in E13.5 yolk sac and labyrinth. These findings taken together suggest that NMHC IIA may play critical roles in the early trophoblast-derived ectoplacental cone and extraembryonic ectoderm, as well as in the yolk sac and labyrinth tissues that form later. Our findings are consistent with phenotypes of constitutive NMIIA knockout mice made earlier, that displayed labyrinth and yolk sac-specific defects, but our findings extend those observations by suggesting possible NMIIA roles in trophoblast lineages as well. These results furthermore demonstrate that K5-Cre gene constructs, previously reported to be activated starting at approximately E12.5 in the forming epidermis, may be widely useful as drivers for activation of cre/lox based gene excision in early embryo extraembronic trophoblast tissues as well.

Aberrant methylation of multiple imprinted genes in embryos of tamoxifen-treated male rats

Genomic imprinting is an epigenetic phenomenon known to regulate fetal growth and development. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased postimplantation loss around mid gestation. Further studies demonstrated the aberrant expression of transcripts of several imprinted genes in the resorbing embryos at days 11 and 13 of gestation including IGF2. In addition, decreased methylation at the Igf2-H19 imprint control region was observed in spermatozoa and in resorbing embryos sired by tamoxifen-treated males. In this study, methylation analysis of the imprinted genes, which were found to be differentially expressed, was done using EpiTYPER in the spermatozoa of tamoxifen-treated rats and in postimplantation embryos sired by tamoxifen-treated rats. Differentially methylated regions (DMRs) for most imprinted genes have not been identified in the rats. Hence, initial experiments were performed to identify the putative DMRs in the genes selected for the study. Increased methylation at CpG islands present in the putative DMRs of a number of imprinted genes was observed in the resorbing embryos sired by tamoxifen-treated male rats. This increase in methylation is associated with the downregulation of most of these genes at the transcript level in resorbing embryos. No change in the methylation status of these genes was observed in spermatozoa. These observations suggest that a deregulation of mechanisms protecting unmethylated alleles from a wave of de novo methylation occurs following implantation.

Clonal Isolation of an Intermediate Pluripotent Stem Cell State

Pluripotent stem cells of different embryonic origin respond to distinct signaling pathways. Embryonic stem cells (ESCs), which are derived from the inner cell mass of preimplantation embryos, are dependent on LIF-Stat3 signaling, while epiblast stem cells (EpiSCs), which are established from postimplantation embryos, require activin-Smad2/3 signaling. Recent studies have revealed heterogeneity of ESCs and the presence of intermediate pluripotent stem cell populations, whose responsiveness to growth factors, gene expression patterns, and associated chromatic signatures are compatible to a state in between ESCs and EpiSCs. However, it remains unknown whether such cell populations represent a stable entity at single-cell level. Here, we describe the identification of clonal stem cells from mouse ESCs with global gene expression profiles representing such a state. These pluripotent stem cells display dual responsiveness to LIF-Stat3 and activin-Smad2/3 at single-cell level and thus named as intermediate epiblast stem cells (IESCs). Furthermore, these cells show accelerated temporal gene expression kinetics during embryoid body differentiation in vitro consistent with a more advanced differentiation stage than that of ESCs. The successful isolation of IESCs supports the notion that traverse from naïve ground state toward lineage commitment occurs gradually in which transition milestones can be captured as clonogenic entity. Our finding provides a new model to better understand the multiple pluripotent states.