Lysophosphatidic Acid Accelerates Bovine In Vitro-Produced Blastocyst Formation through the Hippo/YAP Pathway.

The segregation of trophectoderm (TE) and inner cell mass in early embryos is driven primarily by the transcription factor CDX2. The signals that trigger CDX2 activation are, however, less clear. In mouse embryos, the Hippo-YAP signaling pathway is important for the activation of CDX2 expression; it is less clear whether this relationship is conserved in other mammals. Lysophosphatidic acid (LPA) has been reported to increase YAP levels by inhibiting its degradation. In this study, we cultured bovine embryos in the presence of LPA and examined changes in gene and protein expression. LPA was found to accelerate the onset of blastocyst formation on days 5 and 6, without changing the TE/inner cell mass ratio. We further observed that the expression of TAZ and TEAD4 was up-regulated, and YAP was overexpressed, in LPA-treated day 6 embryos. However, LPA-induced up-regulation of CDX2 expression was only evident in day 8 embryos. Overall, our data suggest that the Hippo signaling pathway is involved in the initiation of bovine blastocyst formation, but does not affect the cell lineage constitution of blastocysts.

Evaluation of in vitro embryotoxicity tests for Chinese herbal medicines.

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.

Effects of MG132 on the in vitro development and epigenetic modification of Debao porcine somatic cell nuclear transfer embryos.

The present study was undertaken to examine the effect of MG132, a proteasome inhibitor, on the in vitro development, zygotic genome activation (ZGA) and epigenetic modification of Debao porcine somatic cell nuclear transfer (SCNT) embryos. Treatment of oocytes with 1 µM MG132 from 30 h to 42 h of maturation and SCNT embryos with 5 µM MG132 for 2 h after fusion resulted in higher blastocyst yield (36.5%) of SCNT embryos compared with the control group (11.0%). The ZGA of SCNT embryos at 2- and 4-cell stages was also enhanced by MG132 treatment through altering the RNA pol II status and increasing the expression of eIF3A and TFIIA. Meanwhile, MG132 treatment also resulted in increase of inner cell mass (ICM) and trophectoderm (TE) and total cell numbers and decrease of apoptotic cell numbers of SCNT blastocysts. Expression of BCL-2, OCT4, NANOG and CDX2 in SCNT blastocysts developed from SCNT embryos and oocytes treated with MG132 was increased significantly (P < 0.01), while the expression of pro-apoptotic BAX gene was suppressed significantly (P < 0.01). In addition, MG132 treatment not only affected the expression patterns of H3K9 acetylation, H3K4 and H3K9 trimethylation, but also regulated the relative expression of SMYD3, ASH2L, KDM5B, HAT1, HDAC1 and HDAC2 of Debao porcine SCNT embryos. These results demonstrate that MG132 treatment can improve the developmental potential of Debao porcine SCNT embryos through regulating the expression of genes related to histone acetylation and the processes of ZGA.

Analysis of Cyclin E1 Functions in Porcine Preimplantation Embryonic Development by Fluorescence Microscopy.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

Direct and Osmolarity-Dependent Effects of Glycine on Preimplantation Bovine Embryos.

Concentrations of glycine (Gly) in embryo culture media are often lower (~0.1 mM) than those in oviductal or uterine fluids (≥1.2 mM). The objective of this study was to determine direct and osmolarity-dependent effects of physiological concentrations of Gly on blastocyst formation and hatching, cell allocation to the trophectoderm (TE) and inner cell mass (ICM), and metabolic activity of bovine embryos. In experiment 1, zygotes were cultured with 100 or 120 mM NaCl and 0 or 1 mM Gly for the first 72 h of culture. Blastocyst formation and hatching were improved (P<0.05) when embryos were cultured with 100 compared to 120 mM NaCl. Inclusion of 1 mM Gly improved (P<0.05) blastocyst formation compared to 0 mM Gly, but this effect was only significant (P<0.05) for embryos cultured with 120 mM NaCl, suggesting bovine embryos can utilize Gly as an osmolyte. In experiment 2, embryos were cultured with 0.1, 1.1, 2.1, or 4.1 mM Gly (100 mM NaCl) for the final 96 h of culture. Blastocyst development was not affected (P>0.05) by Gly, but hatching (0.1 mM Gly, 18.2%) was improved (P<0.05) when embryos were cultured with 1.1 (31.4%) or 2.1 (29.4%) mM Gly. Blastocyst, TE, and ICM cell numbers were not affected (P>0.05) by Gly in either experiment. Blastocysts produced alanine, glutamine, pyruvate, and urea and consumed aspartate, but this metabolic profile was not affected (P>0.05) by Gly. In conclusion, Gly (1.0 mM) improves the development of both early and late stage embryos, but beneficial effects are more pronounced for early embryos exposed to elevated osmolarity.

Effect of growth factors on oocyte maturation and allocations of inner cell mass and trophectoderm cells of cloned bovine embryos.

This study was conducted to determine the additive effects of exogenous growth factors during in vitro oocyte maturation (IVM) and the sequential culture of nuclear transfer (NT) embryos. Oocyte maturation and culture of reconstructed embryos derived from bovine granulosa cells were performed in culture medium supplemented with either epidermal growth factor (EGF) alone or a combination of EGF with insulin-like growth factor-I (IGF-I). The maturation rates of oocytes matured in the presence of EGF or the EGF + IGF-I combination were significantly higher than those of oocytes matured in the presence of only fetal calf serum (FCS) (P 0.05). IGF-I alone or in combination with EGF in sequential embryo culture medium significantly increased the ratio of inner cell mass (ICM) to total blastocyst cells (P < 0.05). Our results showed that the addition of growth factors to IVM and sequential culture media of cloned bovine embryos increased the ICM without changing the total cell number. These unknown and uncontrolled effects of growth factors can alter the allocation of ICM and trophectoderm cells (TE) in NT embryos. A decrease in TE cell numbers could be a reason for developmental abnormalities in embryos in the cloning system.

Signal Inhibition Reveals JAK/STAT3 Pathway as Critical for Bovine Inner Cell Mass Development.

The inner cell mass (ICM) of mammalian blastocysts consists of pluripotent epiblast and hypoblast lineages, which develop into embryonic and extraembryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine Day 8 blastocysts. From the morula stage onward, in vitro fertilized embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signaling pathway components, including TGFbeta1, BMP, EGF, VEGF, PDGF, FGF, cAMP, PI3K, and JAK signals. Using 1) blastocyst quality (by morphological grading), 2) cell numbers (by differential stain), and 3) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR), we identified positive and negative regulators of ICM development and pluripotency. TGFbeta1, BMP, and cAMP and combined VEGF/PDGF/FGF signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF/PDGF/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signaling with AZD1480. This treatment interfered with ICM formation, but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts as well as naive pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3(Y705)) was restricted to ICM nuclei, where it colocalized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3(Y705) signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naive pluripotency markers.

The p38 MAPK signalling pathway is required for glucose metabolism, lineage specification and embryo survival during mouse preimplantation development.

Preimplantation embryo development is an important and unique period and is strictly controlled. This period includes a series of critical events that are regulated by multiple signal-transduction pathways, all of which are crucial in the establishment of a viable pregnancy. The p38 mitogen-activated protein kinase (MAPK) signalling pathway is one of these pathways, and inhibition of its activity during preimplantation development has a deleterious effect. The molecular mechanisms underlying the deleterious effects of p38 MAPK suppression in early embryo development remain unknown. To investigate of the effect of p38 MAPK inhibition on late preimplantation stages in detail, we cultured 2-cell stage embryos in the presence of SB203580 for 48 h and analysed the 8-cell, morula, and blastocyst stages. We determined that prolonged inhibition of the p38 MAPK altered the expression levels of Glut1 and Glut4, decreased glucose uptake during the 8-cell to blastocyst transition, changed the expression levels of transcripts which will be important to lineage commitment, including Oct4/Pou5f1, Nanog, Sox2, and Gata6, and increased cell death in 8-16 cell stage embryos onwards. Strikingly, while the expression levels of Nanog, Gata6 and Oct4/Pou5f1 mRNAs were significantly decreased, Sox2 mRNA was increased in SB203580-treated blastocysts. Taken together, our results provide important insight into the biological processes controlled by the p38 MAPK pathway and its critical role during preimplantation development.

Dysfunction in gap junction intercellular communication induces aberrant behavior of the inner cell mass and frequent collapses of expanded blastocysts in mouse embryos.

PURPOSE: We investigated the role of gap junctions (GJs) in embryological differentiation, and observed the morphological behavior of the inner cell mass (ICM) by time-lapse movie observation (TLM) with gap junction inhibitors (GJis). METHODS: ICR mouse embryos were exposed to two types of GJis in CZB medium: oleamide (0 to 50 µM) and 1-heptanol (0 to 10 mM). We compared the rate of blastocyst formation at embryonic day 4.5 (E4.5) with E5.5. We also observed and evaluated the times from the second cleavage to each embryonic developing stage by TLM. We investigated embryonic distribution of DNA, Nanog protein, and Connexin 43 protein with immunofluorescent staining. RESULTS: In the comparison of E4.5 with E5.5, inhibition of gap junction intercellular communication (GJIC) delayed embryonic blastocyst formation. The times from the second cleavage to blastocyst formation were significantly extended in the GJi-treated embryos (control vs with oleamide, 2224 ± 179 min vs 2354 ± 278 min, p = 0.013). Morphological differences were traced in control versus GJi-treated embryos until the hatching stage. Oleamide induced frequent severe collapses of expanded blastocysts (77.4 % versus 26.3 %, p = 0.0001) and aberrant ICM divisions connected to sticky strands (74.3 % versus 5.3 %, p = 0.0001). Immunofluorescent staining indicated Nanog-positive cells were distributed in each divided ICM. CONCLUSIONS: GJIC plays an important role in blastocyst formation, collapses of expanded blastocysts, and the ICM construction in mouse embryos.

A mRNA landscape of bovine embryos after standard and MAPK-inhibited culture conditions: a comparative analysis.

BACKGROUND: Genes and signalling pathways involved in pluripotency have been studied extensively in mouse and human pre-implantation embryos and embryonic stem (ES) cells. The unsuccessful attempts to generate ES cell lines from other species including cattle suggests that other genes and pathways are involved in maintaining pluripotency in these species. To investigate which genes are involved in bovine pluripotency, expression profiles were generated from morula, blastocyst, trophectoderm and inner cell mass (ICM) samples using microarray analysis. As MAPK inhibition can increase the NANOG/GATA6 ratio in the inner cell mass, additionally blastocysts were cultured in the presence of a MAPK inhibitor and changes in gene expression in the inner cell mass were analysed. RESULTS: Between morula and blastocyst 3,774 genes were differentially expressed and the largest differences were found in blastocyst up-regulated genes. Gene ontology (GO) analysis shows lipid metabolic process as the term most enriched with genes expressed at higher levels in blastocysts. Genes with higher expression levels in morulae were enriched in the RNA processing GO term. Of the 497 differentially expressed genes comparing ICM and TE, the expression of NANOG, SOX2 and POU5F1 was increased in the ICM confirming their evolutionary preserved role in pluripotency. Several genes implicated to be involved in differentiation or fate determination were also expressed at higher levels in the ICM. Genes expressed at higher levels in the ICM were enriched in the RNA splicing and regulation of gene expression GO term. Although NANOG expression was elevated upon MAPK inhibition, SOX2 and POU5F1 expression showed little increase. Expression of other genes in the MAPK pathway including DUSP4 and SPRY4, or influenced by MAPK inhibition such as IFNT, was down-regulated. CONCLUSION: The data obtained from the microarray studies provide further insight in gene expression during bovine embryonic development. They show an expression profile in pluripotent cells that indicates a pluripotent, epiblast-like state. The inability to culture ICM cells as stem cells in the presence of an inhibitor of MAPK activity together with the reported data indicates that MAPK inhibition alone is not sufficient to maintain a pluripotent character in bovine cells.

Functional characterization of SOX2 in bovine preimplantation embryos.

To date, efforts to establish pluripotent embryonic stem cells from bovine embryos have failed. The lack of reliable pluripotency markers is an important drawback when attempting to derive these cells. This study aimed to identify genes upregulated in the inner cell mass (ICM) of bovine blastocysts, and we selected SOX2 for further characterization. Spatial and temporal localization of the SOX2 protein revealed that its expression starts at the 16-cell stage and then becomes restricted to the ICMs of blastocysts. To study the role of SOX2 during the early development of bovine embryos, we designed siRNA to target SOX2. We began by injecting this siRNA into zygotes; the rate at which blastocysts developed declined compared to noninjected or scramble-injected controls. When only one blastomere of a two-cell embryo was injected with SOX2 siRNA, we observed development rates similar to those of controls. Daughter cells of the injected blastomere were tracked by TRITC fluorescence and found to contribute to the ICM, as select cells also lacked SOX2. Gene expression analysis revealed a decrease in SOX2 and NANOG gene expression in siRNA-injected embryos, but OCT4 expression remained unchanged. We conclude that SOX2 localizes exclusively in the ICM of bovine blastocysts, and its downregulation negatively impacts preimplantation development; however, it is still unclear as to why downregulation of SOX2 in one cell of a two-cell embryo does not affect the composition of the ICM.

Regulation of pluripotency of inner cell mass and growth and differentiation of trophectoderm of the bovine embryo by colony stimulating factor 2.

Colony-stimulating factor 2 (CSF2) enhances competence of the bovine embryo to establish and maintain pregnancy after the embryo is transferred into a recipient. Mechanisms involved could include regulation of lineage commitment, growth, or differentiation of the inner cell mass (ICM) and trophectoderm (TE). Experiments were conducted to evaluate regulation by CSF2 of pluripotency of the ICM and differentiation and growth of the TE. Embryos were cultured with 10 ng/ml recombinant bovine CSF2 or a vehicle control from Days 5 to 7 or 6 to 8 postinsemination. CSF2 increased the number of putative zygotes that developed to blastocysts when the percent of embryos becoming blastocysts in the control group was low but decreased blastocyst yield when blastocyst development in controls was high. ICM isolated from blastocysts by lysing the trophectoderm using antibody and complement via immunosurgery were more likely to survive passage when cultured on mitomycin C-treated fetal fibroblasts if derived from blastocysts treated with CSF2 than if from control blastocysts. There was little effect of CSF2 on characteristics of TE outgrowths from blastocysts. The exception was a decrease in outgrowth size for embryos treated with CSF2 from Days 5 to 7 and an increase in expression of CDX2 when treatment was from Days 6 to 8. Expression of the receptor subunit gene CSF2RA increased from the zygote stage to the 9-16 cell stage before decreasing to the blastocyst stage. In contrast, CSF2RB was undetectable at all stages. In conclusion, CSF2 improves competence of the ICM to survive in a pluripotent state and alters TE outgrowths. Actions of CSF2 occur through a signaling pathway that is likely to be independent of CSF2RB.

PDGF signaling is required for primitive endoderm cell survival in the inner cell mass of the mouse blastocyst.

At the end of the preimplantation period, the inner cell mass (ICM) of the mouse blastocyst is composed of two distinct cell lineages, the pluripotent epiblast (EPI) and the primitive endoderm (PrE). The current model for their formation involves initial co-expression of lineage-specific markers followed by mutual-exclusive expression resulting in a salt-and-pepper distribution of lineage precursors within the ICM. Subsequent to lineage commitment, cell rearrangements and selective apoptosis are thought to be key processes driving and refining the emergence of two spatially distinct compartments. Here, we have addressed a role for Platelet Derived Growth Factor (PDGF) signaling in the regulation of programmed cell death during early mouse embryonic development. By combining genetic and pharmacological approaches, we demonstrate that embryos lacking PDGF activity exhibited caspase-dependent selective apoptosis of PrE cells. Modulating PDGF activity did not affect lineage commitment or cell sorting, suggesting that PDGF is involved in the fine-tuning of patterning information. Our results also indicate that PDGF and fibroblast growth factor (FGF) tyrosine kinase receptors exert distinct and non-overlapping functions in PrE formation. Taken together, these data uncover an early role of PDGF signaling in PrE cell survival at the time when PrE and EPI cells are segregated.

The combination of inhibitors of FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human inner cell mass, but does not improve stem cell derivation.

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3ß pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3ß pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3ß pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3ß significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Depletion of Suds3 reveals an essential role in early lineage specification.

Preimplantation development culminates with the emergence of three distinct populations: the inner cell mass, primitive endoderm and trophectoderm. Here, we define the mechanisms underlying the requirement of Suds3 in pre/peri-implantation development. Suds3 knockdown blastocysts exhibit a failure of both trophectoderm proliferation as well as a conspicuous lack of primitive endoderm. Expression of essential lineage factors Nanog, Sox2, Cdx2, Eomes, Elf5 and Sox17 are severely reduced in the absence of Suds3. Importantly, we document deficient FGF4/ERK signaling and show that exogenous FGF4 rescues primitive endoderm formation and trophectoderm proliferation in Suds3 knockdown blastocysts. We also show that Hdac1 knockdown reduces Sox2/FGF4/ERK signaling in blastocysts. Collectively, these data define a role for Suds3 in activation of FGF4/ERK signaling and determine an essential molecular role of Suds3/Sin3/HDAC complexes in lineage specification in vivo.

Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos.

Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.

Embryonic toxicity of sanguinarine through apoptotic processes in mouse blastocysts.

In this study, we examined the cytotoxic effects of sanguinarine, a phytoalexin with antimicrobial, anti-oxidant, anti-inflammatory and pro-apoptotic effects, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation via embryo transfer. Blastocysts treated with 0.5-2 µM sanguinarine exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with sanguinarine were lower than that of their control counterparts. Moreover, in vitro treatment with 0.5-2 µM sanguinarine was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that sanguinarine induces apoptosis and retards early post-implantation development in vitro and in vivo. In addition, sanguinarine induces apoptotic injury effects on mouse blastocysts through intrinsic and extrinsic apoptotic signaling processes to impair sequent embryonic development. However, the extent to which sanguinarine exerts teratogenic effects on early human development is not known at present, and further studies are required to establish effective protection strategies against its cytotoxic effects.

3,4-Dihydroxyflavone acts as an antioxidant and antiapoptotic agent to support bovine embryo development in vitro.

The effects of two antioxidants, superoxide dismutase (SOD) and the flavonoid 3,4-dihydroxyflavone (DHF), on bovine embryo development in vitro were examined. Blastocyst development, total cell and inner cell mass (ICM) numbers, intracellular levels of reactive oxygen species (ROS), apoptotic indices and gene expression levels were examined before and after treatment of day 2 bovine embryos (≥2-4 cells) with various concentrations of 3,4-DHF or SOD for 6 days. Statistical analysis was performed using analysis of variance, with significance defined at the P<0.05 level. SOD had no significant effect on bovine embryo development at any tested concentration (control, 32.8%; 300 U/ml, 33.9%; 600 U/ml, 24.2%). In contrast, 10 µM 3,4-DHF promoted higher blastocyst development (39.3%) than any other concentration (control, 26.7%; 1 µM, 30.3%; 50 µM, 29.5%; 100 µM, 20.5%). Compared with 300 U/ml SOD, 10 µM 3,4-DHF resulted in significantly higher blastocyst development (44.2%) (control, 31.5%; SOD 300 U/ml, 33.6%). Treatment with 3,4-DHF increased the ICM cell number and reduced intracellular ROS production and apoptotic cell numbers. When O(2) tension was decreased from 20% (high tension) to 5% (low tension), embryo development rates were doubled regardless of 3,4-DHF treatment. Under high O(2) tension, 10 µM 3,4-DHF treatment may render bovine embryo development similar to a low O(2) tension environment. The best blastocyst development was obtained under low O(2) tension plus 10 µM 3,4-DHF treatment. The relative expression levels of antioxidant (MnSOD), antiapoptotic (Survivin, Bax inhibitor) and growth-related genes (IFN-τ, Glut-5) were significantly increased after 3,4-DHF treatment, while the expression levels of oxidant (Sox) and apoptotic genes (Caspase-3 and Bax) were reduced. These results suggest that 3,4-DHF may promote the in vitro development of bovine embryos through its antioxidant and antiapoptotic effects.

An FGF4-FRS2alpha-Cdx2 axis in trophoblast stem cells induces Bmp4 to regulate proper growth of early mouse embryos.

A variety of stem cells are controlled by the actions of multiple growth factors in vitro. However, it remains largely unclear how growth factors control the proliferation and differentiation of stem cells in vivo. Here, we describe a novel paracrine mechanism for regulating a stem cell niche in early mammalian embryos, which involves communication between the inner cell mass (ICM) and the trophectoderm, from which embryonic stem (ES) cells and trophoblast stem (TS) cells can be derived, respectively. It is known that ES cells produce fibroblast growth factor (FGF)4 and that TS cells produce bone morphogenetic protein (Bmp)4. We provide evidence that FRS2alpha mediates activation of the extracellular signal-regulated progein kinase (ERK) pathway to enhance expression of transcription factor Cdx2 in TS cells in response to FGF4. Cdx2 in turn binds to an FGF4-responsive enhancer element of the promoter region of Bmp4, leading to production and secretion of Bmp4. Moreover, exogenous Bmp4 is able to rescue the defective growth of Frs2alpha-null ICM. These findings suggest an important role of Cdx2 for production of Bmp4 in TS cells to promote the proper growth of early mouse embryos.

Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo.

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naïve epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naïve epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.