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The recombinantelastin engineering bacteria was constructed by genetic engineering technology, and then recombinant elastin was obtained by fermentation purification technology. The recombinante lastin was characterized and identified by N-terminal sequencing of amino acids, molecular wght determination, analysis of amino acid composition, protein purity analysis, endotoxicity determination, infrared spectroscopy and circular dichroism analysis. To explore the roles of recombiant elastin in UVB-induced skin photoaging, the effects of recombinant elastin on the adhesion ability and cell viability of L929 mouse embryonic fibroblasts, as well as the effects of recombinant elastin on the expression levels of Collagen, Collagen, Elastin and matrix metalloproteinase 1 MMP-1, were investigated in the model of UVB-induced L929 cell photoaging. The results showed that the secondary structure of recombinant elastin was similar to that of natural elastin. The recombinant elastin showed certain cell viability and no cytotoxicity at 0.1 mg/mL, which significantly promoted the adhesion and proliferation of L929 cells. Additionally, recombinant elastin could also improve the mRNA transcription levels of Collagen, Collagen and Elastin in UVB irradiated cells by down-regulating the expression of MMP-1 in UVB irradiated cells, thus protecting L929 cells from light damage. That was why recombinant elastin could improve the survival rate of photodamaged fibroblasts. [ABSTRACT FROM AUTHOR]

During mouse pre-implantation development, the formation of the blastocoel, a fluid-filled lumen, breaks the radial symmetry of the blastocyst. The factors that control the formation and positioning of this basolateral lumen remain obscure. We found that accumulation of pressurized fluid fractures cell-cell contacts into hundreds of micrometer-size lumens. These microlumens eventually discharge their volumes into a single dominant lumen, which we model as a process akin to Ostwald ripening, underlying the coarsening of foams. Using chimeric mutant embryos, we tuned the hydraulic fracturing of cell-cell contacts and steered the coarsening of microlumens, allowing us to successfully manipulate the final position of the lumen. We conclude that hydraulic fracturing of cell-cell contacts followed by contractility-directed coarsening of microlumens sets the first axis of symmetry of the mouse embryo. [ABSTRACT FROM AUTHOR]

Embryo vitrification has advantages in assisted reproduction yet it also induces zona hardening. Laser zona thinning (LZT) is considered as a solution yet its efficacy and security have not been well studied. In this study, we used vitrified-warmed morulae from 2-month-old and 10-month-old ICR female mice as model to investigate the impacts that LZT treatment brings to the in vitro hatching process and implantation by analyzing hatching rate, implantation rate, and blastocyst quality. The results showed that the fully hatched rate was significantly higher after LZT treatment for both young (25.7% vs. 16.2%, P < 0.05) and aged (36.6% vs. 13.2%, P < 0.01) mice. For zona-thinned morulae in young mice, its onset of hatching occurred earlier (28.6% vs. 8.8%, P < 0.01) at D4 and with a greater percentage of U-shaped hatching at D5 (48.3% vs. 33.0%, P < 0.05). LZT treatment did not induce expression change of apoptosis-related genes in all groups (P > 0.05), but for young mice, the total cell number of day 5 blastocyst in zona-thinned group was significantly less than that of the control group (40.6 ± 5.1 vs. 59.9 ± 14.5, P < 0.01). At last, there was an increasing implantation rate in zona-thinned compared to the control group for young (63.8% vs. 52.5%, P > 0.05) and aged (55.6% vs. 47.2%; P > 0.05) mice after embryos were bilaterally transferred in the same recipient. In conclusion, the significant increase of fully hatched rate after LZT treatment is related to the advanced onset of hatching as well as the enhancement of superior hatching structure, and LZT also lead to a better implantation after embryo transfer. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) have been implicated in numerous biological events. However, to date, the role of HDAC6 in early embryos remains unknown. In the current study, Tubastatin A (TubA), a potent HDAC6 inhibitor, was used to block HDAC6 activity in mouse embryos. We found that TubA exposure significantly reduced the blastocyst formation of early embryos. Confocal microscopy revealed the markedly increased chromosomal congression failure in the mouse embryos treated with the HDAC6 inhibitor. Moreover, the HDAC6 inhibition resulted in the overproduction of reactive oxygen species (ROS) in embryos. In addition, we observed the accumulation of phosphorylated γH2AX in TubA‐treated embryos, indicative of the increased DNA damage. In line with this, cell apoptosis of blastocysts was frequently detected in HDAC6‐deficient embryos compared with their controls. Altogether, our data indicate that HDAC6 may serve as an important regulator of chromatin structure and mitochondrial function, determining the developmental potential of the early embryos of mouse. Our data indicate that histone deacetylases 6 (HDAC6) may serve as an important regulator of chromatin structure and mitochondrial function, determining the developmental potential of the early embryos of mouse. [ABSTRACT FROM AUTHOR]

Cumulus–oocyte complex (COC) expansion is essential for ovulation and fertilisation and is linked to oocyte quality. Hyaluronan (HA), the major matrix constituent, is cross-linked via inter-α-inhibitor heavy chains (HCs), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene 6 (TSG-6). All except HCs are secreted by cumulus cells in response to oocyte-secreted factors, which signal via SMAD pathways. The double mutant (DM) mouse generates oocytes lacking complex N- and O-glycans due to oocyte-specific deletion of core 1 β1,3-galactosyltransferase (C1galt1) and N-acetylglucosaminyltransferase I (Mgat1) and has modified cumulus expansion. We compared COCs before expansion (48 h-post-pregnant mare serum gonadotrophin (PMSG)) and at late-stage expansion (9 h-post-human chorionic gonadotrophin (hCG); control n = 3 mice, DM n = 3 per group). Using histochemistry the levels of HA, HCs, PTX3, TSG-6 and phosphorylated-SMAD1/5/8 and -SMAD2 (12–25 COCs per group) were assessed. DM COCs did not differ from Controls in cumulus size or cell density at 9 h-post-hCG; however, HA and HC levels and phosphorylated-SMAD1/5/8 were reduced. Furthermore, no correlations were found between the levels of matrix molecules and cumulus area in DM or Control samples. These data suggest that HA and HCs can support cumulus expansion provided that they are present above minimum threshold levels. We propose that oocyte-specific ablation of C1galt1 and Mgat1 may affect bone morphogenetic protein 15 synthesis or bioactivity, thereby reducing SMAD1/5/8 phosphorylation and HA production. Cumulus-–oocyte complex expansion is essential for ovulation and fertilisation. This study revealed that cumulus area is not altered in mice with oocyte-specific deletion of C1galt1 and Mgat1 , despite a reduction in hyaluronan and other molecules associated with cumulus expansion. A minimum threshold may exist for supporting cumulus expansion. Oocyte-secreted factors may be affected by the deletions, causing altered cumulus composition. [ABSTRACT FROM AUTHOR]

Abstract In 1972, a procedure was derived to cryopreserve mouse embryos. Over the past four decades, this procedure has been adapted to freeze embryos of more than twenty-five mammalian species. Cryopreservation of embryos has become a routine procedure in both veterinary and human medicine, having been used to freeze millions of embryos of mice and cattle, and many hundreds of thousands of human embryos. After transfer into appropriate foster mothers, cryopreserved embryos have developed into innumerable live offspring. This article describes the background that led to the derivation of the procedure and the events that transpired during its development. The first successful embryo cryopreservation procedure was developed by collaboration of three investigators, each bringing a special expertise and perspective to the project. [ABSTRACT FROM AUTHOR]

Abstract At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection. [ABSTRACT FROM AUTHOR]

Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid family of compounds. Due to the presence of strong carbon–fluorine bonds, it is practically nonbiodegradable and highly persistent in the environment. PFOA has been detected in the follicular fluid of women, and positively associated with reduced fecundability and infertility. However, there are no reports concerning the experimental evaluation of PFOA on oocyte toxicity in mammals. The aim of the present study was to determine if PFOA is able to induce oxidative stress in fetal ovaries and cause apoptosis in oocytes in vitro. In addition, since inhibition of the gap junction intercellular communication (GJIC) by PFOA has been demonstrated in liver cells in vivo and in vitro, the effect of PFOA on the GJIC between the oocyte and its supportive cumulus cells was studied. Results show that PFOA induced oocyte apoptosis and necrosis in vitro (medium lethal concentration, LC50 = 112.8 μM), as evaluated with Annexin‐V‐Alexa 508 in combination with BOBO‐1 staining. Reactive oxygen species (ROS) levels, as assessed by DCFH‐DA, increased significantly in fetal ovaries exposed to ¼ LC50 (28.2 μM, a noncytotoxic and relevant occupational exposure concentration) and LC50 PFOA ex vivo. This perfluorinated compound also caused the blockage of GJIC in cumulus cells‐oocyte complexes (COCs) obtained from female mice exposed in vivo, as evaluated by calcein transfer from cumulus cells to the oocyte. The ability of PFOA of disrupting the GJIC in COCs, generating ROS in the fetal ovary and causing apoptosis and necrosis in mammal's oocytes, might account for the reported association between increasing maternal plasma concentrations of PFOA with reduced fertility in women. [ABSTRACT FROM AUTHOR]

Enniatins are mycotoxins of Fusarium fungi that naturally exist as mixtures of cyclic depsipeptides. Previous reports have documented hazardous effects of enniatins on cells, such as apoptosis. However, their effects on pre‐ and post‐implantation embryonic development require further clarification. Here, we showed for the first time that enniatin B1 (ENN B1) exerts cytotoxic effects on mouse blastocyst‐stage embryos and induces intracellular oxidative stress and immunotoxicity in mouse fetuses. Co‐incubation of blastocysts with ENN B1 triggered significant apoptosis and led to a decrease in total cell number predominantly through loss of inner cell mass. In addition, ENN B1 appeared to exert hazardous effects on pre and postimplantation embryo development potential in an in vitro development assay. Treatment of blastocysts with 1‐10 μM ENN B1 led to increased resorption of post‐implantation embryos and decreased fetal weight in the embryo transfer assay in a dose‐dependent manner. Importantly, in an in vivo model, intravenous injection with ENN B1 (1, 3, and 5 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst‐stage embryos and impairment of embryonic development from the zygote to blastocyst stage, subsequent degradation of embryos, and further decrease in fetal weight. Intravenous injection with 5 mg/kg body weight/d ENN B1 additionally induced a significant increase in total reactive oxygen species (ROS) content and transcription levels of genes encoding antioxidant proteins in mouse fetal liver. Moreover, ENN B1 triggered apoptosis through ROS generation and strategies to prevent apoptotic processes effectively rescued ENN B1‐mediated hazardous effects on embryonic development. Transcription levels of CXCL1, IL‐1β, and IL‐8 related to innate immunity were downregulated after intravenous injection of ENN B1. These results collectively highlight the potential of ENN B1 to exert cytotoxic effects on embryos as well as oxidative stress and immunotoxicity during mouse embryo development. [ABSTRACT FROM AUTHOR]

The recent advance of technologies enables us to trace the cell fate in vivo by marking the cells that express the gene of interest or by barcoding them at a single cell level. Various tamoxifen-inducible Cre-recombinase mice combined with Rosa-floxed lines are utilized. In this review, with the results revealed by lineage tracing assays, we re-visit the long-standing debate for the origin of hematopoietic stem cells in the mouse embryo, and introduce the view of native hematopoiesis, and possible leukemic-initiating cells emerged during fetal stages. [ABSTRACT FROM AUTHOR]

Abstract Stasimon (also known as Tmem41b) is an evolutionarily conserved transmembrane protein first identified for its contribution to motor system dysfunction in animal models of the childhood neurodegenerative disease spinal muscular atrophy (SMA). Stasimon was shown to be required for normal neurotransmission in the motor circuit of Drosophila larvae and proper development of motor axons in zebrafish embryos as well as to suppress analogous neuronal phenotypes in SMA models of these organisms. However, the subcellular localization and molecular functions of Stasimon are poorly understood. Here, we combined immunoprecipitation with mass spectrometry to characterize the Stasimon interactome in mammalian cells, which reveals association with components of the endoplasmic reticulum (ER), mitochondria, and the COPI vesicle trafficking machinery. Expanding on the interaction results, we used subcellular fractionation studies and super-resolution microscopy to identify Stasimon as an ER-resident protein that localizes at mitochondria-associated ER membranes (MAM), functionally specialized contact sites between ER and mitochondria membranes. Lastly, through characterization of novel knockout mice, we show that Stasimon is an essential gene for mouse embryonic development. Together, these findings identify Stasimon as a novel transmembrane protein component of the MAM with an essential requirement for mammalian development. Highlights • Stasimon associates with ER, mitochondria, and COPI vesicle trafficking machinery. • Stasimon localizes at mitochondria-associated ER membranes. • Stasimon is essential for mouse embryonic development. [ABSTRACT FROM AUTHOR]

Abstract This study was conducted to compare the effect of minimum volume Spatula MVD vitrification (VIT) versus traditional slow freezing (SLF) of mouse embryos. A total of 2,617 8-cell in vivo derived and 2-cell in vitro produced B6D2 mouse embryos were subjected to freezing/thawing or vitrification/warming, while fresh embryos were used as control group. Embryo recovery, survival and development rate, pregnancy rate and offspring production were analyzed. In Experiment 1, 8-cell in vivo derived embryos were subjected to in vitro culture, resulting in greater survival and development rates at 3.5 days post coitum stage in VIT than in SLF group (P < 0.05). Although both methods reached an acceptable hatching rate (41.0% and 49.7% for VIT and SLF, respectively; P=NS), it was significantly lower respect to the control group (67.8%, P < 0.01). In Experiment 2, 2-cell in vitro produced mouse embryos showed a similar recovery rate from the device after freezing/thawing or vitrification/warming (∼84%), however survival rate was significantly higher for vitrified/warmed (94.7%) than frozen/thawed embryos (85.1%; P < 0.01). Vitrified/warmed and control fresh embryos were transferred to surrogate mothers, revealing no differences both in pregnancy and offspring production rates. Our data demonstrate that minimum volume Spatula MVD method is a simple home-made useful technique for vitrification of 2-cell and 8-cell mouse embryos produced either in vitro or in vivo. [ABSTRACT FROM AUTHOR]

Highlights • We combined assays using mouse embryonic stem cells (mESCs) and zebrafish embryos to evaluate developmental toxicity of 11 chemicals. • Using mESCs, proliferation, differentiation, and cytotoxicity of the chemicals were measured. • In zebrafish embryos, lethality and the lowest effect level concentrations for morphological malformations were determined. • The combined mESC and zebrafish toxicity data of the chemicals correlated well to rodent in vivo toxicity (R = 0.88, P < 0.001). Abstract The assays in this study utilize mouse embryonic stem cells (mESCs) and zebrafish embryos to evaluate the potential developmental toxicity of industrial and pharmaceutical chemicals. A set of eleven chemicals of known mammalian in vivo teratogenicity were tested in the assays and correlations to mammalian data. Using mESCs, proliferation, differentiation, and cytotoxicity of the chemicals were measured. In zebrafish embryos, lethality and the lowest effect level concentrations for morphological malformations were determined. Clustering of the assays based on frequency of affected assays resulted in a ranking of the test compounds that correlated to in vivo rodent data (R = 0.88, P < 0.001). We conclude that the combination of ESC- and zebrafish-based assays provides a valuable platform for the prioritization of pharmaceutical and industrial chemicals for further testing of developmental toxicity in rodents. [ABSTRACT FROM AUTHOR]

In the field of X-ray microcomputed tomography (μCT) there is a growing need to reduce acquisition times at high spatial resolution (approximate micrometers) to facilitate in vivo and high-throughput operations. The state of the art represented by synchrotron light sources is not practical for certain applications, and therefore the development of high-brightness laboratoryscale sources is crucial. We present here imaging of a fixed embryonic mouse sample using a compact laser-plasma-based X-ray light source and compare the results to images obtained using a commercial X-ray μCT scanner. The radiation is generated by the betatron motion of electrons inside a dilute and transient plasma, which circumvents the flux limitations imposed by the solid or liquid anodes used in conventional electron-impact X-ray tubes. This X-ray source is pulsed (duration <30 fs), bright (>1010 photons per pulse), small (diameter <1 μm), and has a critical energy >15 keV. Stable X-ray performance enabled tomographic imaging of equivalent quality to that of the μCT scanner, an important confirmation of the suitability of the laserdriven source for applications. The X-ray flux achievable with this approach scales with the laser repetition rate without compromising the source size, which will allow the recording of high-resolution μCT scans in minutes. [ABSTRACT FROM AUTHOR]

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for de novo purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene Mthfd1l encoding methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by Mthfd1l deletion are not known. Here, we performed untargeted metabolomics on whole Mthfd1l-null and wildtype mouse embryos in combination with isotope tracer analysis in mouse embryonic fibroblast (MEF) cell lines to identify Mthfd1l deletion-induced disruptions in 1C metabolism, glycolysis, and the TCA cycle. We found that maternal formate supplementation largely corrects these disruptions in Mthfd1l-null embryos. Serine tracer experiments revealed that Mthfd1l-null MEFs have altered methionine synthesis, indicating that Mthfd1l deletion impairs the methyl cycle. Supplementation of Mthfd1l-null MEFs with formate, hypoxanthine, or combined hypoxanthine and thymidine restored their growth to wildtype levels. Thymidine addition alone was ineffective, suggesting a purine synthesis defect in Mthfd1l-null MEFs. Tracer experiments also revealed lower proportions of labeled hypoxanthine and inosine monophosphate in Mthfd1l-null than in wildtype MEFs, suggesting that Mthfd1l deletion results in increased reliance on the purine salvage pathway. These results indicate that disruptions of mitochondrial 1C metabolism have wide-ranging consequences for many metabolic processes, including those that may not directly interact with 1C metabolism. [ABSTRACT FROM AUTHOR]

GABAergic interneurons (GABA, g-aminobutyric acid) regulate neural-circuit activity in the mammalian cerebral cortex. These cortical interneurons are structurally and functionally diverse. Here, we use single-cell transcriptomics to study the origins of this diversity in the mouse. We identify distinct types of progenitor cells and newborn neurons in the ganglionic eminences, the embryonic proliferative regions that give rise to cortical interneurons. These embryonic precursors show temporally and spatially restricted transcriptional patterns that lead to different classes of interneurons in the adult cerebral cortex. Our findings suggest that shortly after the interneurons become postmitotic, their diversity is already patent in their diverse transcriptional programs, which subsequently guide further differentiation in the developing cortex. [ABSTRACT FROM AUTHOR]

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro . Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes. [ABSTRACT FROM AUTHOR]

Early life nutritional deficiencies can lead to increased cardiovascular susceptibility to environmental exposures. Thus, the purpose of this study was to examine the effect of early life persistent vitamin D deficiency (VDD) on the cardiopulmonary response to a particulate matter-enhanced photochemical smog. Mice were fed a VDD or normal diet (ND) after weaning. At 17 weeks of age, mice were implanted with radiotelemeters to monitor electrocardiogram, heart rate (HR), and heart rate variability (HRV). Ventilatory function was measured throughout the diet before and after smog exposure using whole-body plethysmography. VDD mice had lower HR, increased HRV, and decreased tidal volume compared with ND. Regardless of diet, HR decreased during air exposure; this response was blunted by smog in ND mice and to a lesser degree in VDD. When compared with ND, VDD increased HRV during air exposure and more so with smog. However, smog only increased cardiac arrhythmias in ND mice. This study demonstrates that VDD alters the cardiopulmonary response to smog, highlighting the possible influence of nutritional factors in determining responses to air pollution. 1 he mechanism of how VDD induces these effects is currently unknown, but modifiable factors should be considered when performing risk assessment of complex air pollution atmospheres. [ABSTRACT FROM AUTHOR]

Although transplantation of stem cells improves recovery of the nervous tissue, little is known about the influence of different brain regions on transplanted cells. After we confirmed that cells with uniform differentiation potential can be generated in independent experiments, one million of neural stem cells isolated from B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse embryos were transplanted into the brain 24 h after induction of stroke. The lateral ventricles, the corpus callosum and the striatum were tested. Two and four weeks after the transplantation, the cells transplanted in all three regions have been attracted to the ischemic core. The largest number of attracted cells has been observed after transplantation into the striatum. Their differentiation pattern and expression of neuroligin 1, SynCAM 1, postsynaptic density protein 95 and synapsin 1 followed the same pattern observed during in vitro cultivation and it did not differ among the tested regions. Differentiation pattern of the cells transplanted in the stroke-affected and healthy animals was the same. On the other hand, neural stem cells transplanted in the striatum of the animals affected by stroke exhibited significantly increased survival rates reaching 260 ± 19%, when compared to cells transplanted in their wild type controls. Surprisingly, improved survival two and four weeks after transplantation was not due to increased proliferation of the grafted cells and it was accompanied by decreased levels of activity of Casp3 (19.56 ± 3.1% in the stroke-affected vs. 30.14 ± 2.4% in healthy animals after four weeks). We assume that the decreased levels of Casp3 in cells transplanted near the ischemic region was linked to increased vasculogenesis, synaptogenesis, astrocytosis and axonogenesis detected in the host tissue affected by ischemia. [ABSTRACT FROM AUTHOR]

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5 days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel ( I f ), as well as L-type Ca 2+ channel ( I CaL ), and sodium channel ( I Na ). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Maintaining mouse stocks as frozen materials offers both ethical and economical advantages over live animal breeding if the lines are not actively used. The European Mouse Mutant Archive (EMMA) promotes the archiving and distribution of important mouse models for biomedical research through the cryopreservation of their embryos and gametes. Embryo freezing in liquid nitrogen (LN 2 ) at −196 °C has traditionally been the method of choice for archiving mouse lines. However, sperm freezing is emerging as a more convenient alternative due to the application of innovative cryopreservation and recovery protocols. In addition, frozen spermatozoa are less sensitive to post-freezing temperature fluctuations. We have previously reported that spermatozoa frozen using standard laboratory protocols can be safely stored in a −80 °C freezer or in dry ice (−79 °C) for at least seven days. We now report the extension of this period of maintenance at −80 °C up to two 2 years both for wild type and mutant strains, indicating that once frozen, mouse spermatozoa are quite resistant and can be transported, shared and stored at −80 °C for a long time without a significant loss of viability. The importance of this finding will be especially relevant for small laboratories with no constant access to liquid nitrogen and for labs generating many mouse mutant lines by CRISPR/Cas9 who do not want to saturate the limited space of a LN 2 tank, using a more accessible −80 °C freezer. [ABSTRACT FROM AUTHOR]

Abstract: Aim: We investigated whether recombinant mouse interleukin‐6 (IL‐6) affects the development of preimplantation embryos and induces the ‐signal transducers and activators of transcription (JAK–STAT) signaling pathway by binding IL‐6 signal transducer (IL‐6st) and regulating Fos and Jun gene expression, thereby accounting for the negative effect of superovulation on embryo development. Methods: We compared rates of blastocyst formation from embryos cultured with different concentrations of IL‐6 or/and anti‐interleukin 6 receptor antibody (anti‐IL‐6RAb) in superovulated experimental and normal control groups. IL‐6 expression in preimplantation embryos was determined by immunofluorescence identification. Fos, Jun and IL‐6st messenger RNA expression was detected by PCR and microarray experiments. Results: Rates of blastocyst formation significantly decreased in superovulated embryos, whether or not they were incubated in 0.1, 1, 25 or 50 pg/mL IL‐6, (P < 0.01) compared to embryos from naturally ovulated controls, whereas incubation in 5 and 10 pg/mL IL‐6 reversed the negative effects of superovulation. The addition of anti‐IL‐6RAb to naturally ovulated embryos reduced blastocyst rates to those of superovulated embryos. Gene chip analysis indicated that the JAK–STAT signaling pathway contained differentially expressed IL‐6, IL‐6st, Jun and Fos genes. Both anti‐IL‐6RAb or ovarian stimulation downregulated IL‐6st, Jun, and Fos messenger RNA expression, but expression of the same three genes increased in 5 pg/mL IL‐6. Conclusion: Ovarian stimulation negatively impacts the development of preimplantation embryos by reducing IL‐6 release. IL‐6 affects the rate of development of zygotes to blastocyst by regulating IL‐6st, Fos and Jun expression in the JAK–STAT signaling pathway. [ABSTRACT FROM AUTHOR]

Background: Quercetin is a flavonoid with the ability to improve the growth of embryos in vitro, and actinomycin D is an inducer of apoptosis in embryonic cells. Objective: The aim was to evaluate the effect of quercetin on the number of viable and apoptotic cells, the zona pellucida (ZP) thickness and the hatching rate of preimplantation embryos exposed to actinomycin D in mice. Materials and Methods: Two-cell embryos were randomly divided into four groups (Control, Quercetin, actinomycin D, and Quercetin + actinomycin D group). Blastocysts percentage, hatched blastocysts, and ZP thickness of blastocysts was measured. The number of blastomeres was counted by Hoechst and propidium iodide staining and the apoptotic cells number was counted by TUNEL assay. Results: The results showed that the use of quercetin significantly improved the growth of embryos compared to the control group (p=0.037). Moreover, quercetin reduced the destructive effects of actinomycin D on the growth of embryos significantly (p=0.026). Conclusion: quercetin may protect the embryos against actinomycin D so that increases the number of viable cells and decreases the number of apoptotic cells, which can help the expansion of the blastocysts, thinning of the ZP thickness and increasing the hatching rate in mouse embryos. [ABSTRACT FROM AUTHOR]

"The Atlas of Mouse Development" by Kaufman is a classic paper atlas that is the de facto standard for the definition of mouse embryo anatomy in the context of standard histological images. We have redigitized the original haematoxylin and eosin-stained tissue sections used for the book at high resolution and transferred the hand-drawn annotations to digital form. We have augmented the annotations with standard ontological assignments (EMAPA anatomy) and made the data freely available via an online viewer (eHistology) and from the University of Edinburgh DataShare archive. The dataset captures and preserves the definitive anatomical knowledge of the original atlas, provides a core image set for deeper community annotation and teaching, and delivers a unique high-quality set of high-resolution histological images through mammalian development for manual and automated analysis. [ABSTRACT FROM AUTHOR]

The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age. [ABSTRACT FROM AUTHOR]

Quercetin is a natural flavonoid with strong antioxidant activity. In the present study, we evaluate the influence of different concentrations of quercetin (QT) on intracytoplasmic oxidative stress and glutathione (GSH) concentration, during in vitro maturation (IVM) and fertilization in mouse oocytes. IVM was carried out in the presence of control (QT0), 5 (QT5), 10 (QT10), and 20 (QT20) µg/mL of QT. Nuclear maturation, intracellular GSH and ROS content were evaluated following the IVM. In these oocytes, we subsequently evaluated the effect of QT supplementation on embryo development, including 2-cell, 8-cell, and blastocyst rate. The results of the present study showed that the supplementation of 10 µg/mL QT in maturation medium increased the number of MII oocytes. In addition, fertilization and blastocyst rate in QT10 treatment group were significantly higher in comparison to the other groups, and elevated the amount of intracellular GSH content compared to other QT concentrations and control groups. The intracellular ROS level was the lowest among oocytes matured in Q5 and Q10 treatment groups. This result suggested that quercetin dose-dependently improves nuclear maturation and embryo development, via reducing intracytoplasmic oxidative stress in mature oocyte. [ABSTRACT FROM AUTHOR]

Although lipid phase transition is believed to be among the major damaging factors in oocytes and preimplantation embryos cryopreservation, lack of the appropriate experimental methods limits investigation of this phenomenon. Herein, we demonstrate the capabilities of Raman spectroscopy to detect the lipid phase transition within the freezing preimplantation mouse embryos. We exploit the sensibility of antisymmetric CH 2 Raman peak to the phase state of lipids. It is shown that during the freezing of the mouse embryos the lipid phase transition occurs at the temperatures between −7 and 0 °C. Similar temperature dependences of CH 2 mode intensities are found for lipids in the preimplantation embryos and a 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, implying the similarity in the occupation rules of conformational states. Raman spectroscopy is considered as a method of choice to study the lipid phase transition during preimplantation mammalian embryos freezing and cryopreservation. [ABSTRACT FROM AUTHOR]

The potential toxicity of neonicotinoids (thiacloprid, acetamiprid, thiamethoxam and clothianidin) as well as related commercial products Calypso 480SC (thiacloprid mixture), Mospilan 20SP (acetamiprid mixture) and Agita 10WG (thiamethoxam mixture) on developmental capacities and quality of preimplantation embryos was evaluated. During in vitro tests, isolated 2-cell stage mice embryos were cultured in media with various concentrations of active compounds or commercial products until blastocyst formation. As found using stereomicroscopic examination, all neonicotinoids at highest (100 μM) concentration negatively affected embryonic development (P < 0.001). Fluorescence staining revealed that the blastocysts obtained displayed lower numbers of blastomeres and elevated incidence of cell death. Thiacloprid and acetamiprid decreased quality of blastocysts also at 10 μM concentration. From the tested products only Calypso 480SC containing 10 μM of thiacloprid showed harmful impact on embryo quality. In an experiment using rabbit embryos, similar negative effect of thiacloprid in vitro was recorded. In vivo testing confirmed that blastocysts collected from thiacloprid-treated mice displayed lower total cell counts than blastocysts from controls. The sensitivity of embryonic cells to neonicotinoids is in the order of thiacloprid > acetamiprid, thiomethoxam > clothianidin. Thiacloprid impairs development and quality of both mouse and rabbit preimplantation embryos, and shows embryotoxicity even at acute reference dose. [ABSTRACT FROM AUTHOR]

Surgical transfer of embryos is carried out daily in animal facilities worldwide for the rederivation of mouse strains/lines, among other purposes. Current protocols described in laboratory manuals recommend using a high number of embryos during transfer, typically in the range of 15 up to 25. To optimize the use of resources it is necessary to estimate and relate the effort required and the yield obtained. Here, we analyse the balance between the number of embryos transferred (the effort), and the yield as the number of born pups obtained from surgical embryo transfer. To accomplish this, we have analyzed data obtained during rederivation of nearly one hundred lines of mice to a new animal facility. Our results confirm that the use of increasing numbers of embryos per transfer increases the yields of born pups, as has been described previously in the literature, but they also highlight the disproportionate effort required, i.e. in the number of embryos that needed to be transferred. An estimate of the mean expected yields of surgical transfers and their comparison with the actual observed yields indicated that the balance between effort and yield is optimized when using lower numbers of embryos than in currently used protocols, in the range of 8 to 12. Given the heterogeneous nature of the data presented and analyzed here, which is from a population of mice that may be considered as representative of any animal facility, our optimization approach should help save resources in similar facilities and improve the yields of embryo transfer procedures. [ABSTRACT FROM AUTHOR]

It has been suggested that first embryo cleavage can be related with the embryonic–abembryonic axis at blastocyst stage in mice. Thus, cells of the 2-cell embryo might be already biased to form the inner cell mass or trophectoderm. This study was conducted to observe the possible effects of embryo biopsy on cell allocation patterns during embryo preimplantation in two different mouse strains and the effects of these patterns on further development. First, one blastomere of the 2-cell embryo was injected with a lipophilic tracer and cell allocation patterns were observed at blastocyst stage. Blastocysts were classified into orthogonal, deviant or random pattern. For the first experiment, embryos were biopsied at 8-cell stage and total cell counts (TCC) were annotated. Furthermore, non-biopsied blastocysts were transferred into foster mothers. Then, pups and their organs were weighed two weeks after birth. Random pattern was significantly recurrent (≈60%), against orthogonal (<22%) and deviant (<22%) patterns among groups. These patterns were not affected by biopsy procedure. However, TCC on deviant embryos were reduced after biopsy. Moreover, no differences were found between patterns for implantation rates, litter size, live offspring and organ weights (lungs, liver, pancreas and spleen). However, deviant pups presented heavier hearts and orthogonal pups presented lighter kidneys among the group. In conclusion, these results suggest that single blastomere removal does not disturb cell allocation patterns during preimplantation. Nonetheless, the results suggest that embryos following different cell allocation patterns present different coping mechanisms against in vitro manipulations and further development might be altered. [ABSTRACT FROM AUTHOR]

Antioxidant of bamboo leaves (AOB) was certified to be a natural antioxidant by the Chinese Ministry of Health in 2003. However, the effects of AOB on animal reproductive and developmental functions remain unclear. The present study aimed to investigate the effects of different concentrations of AOB on mouse embryonic fibroblast (MEF) cells, and to examine the underlying molecular mechanism through which AOB affects the proliferation and apoptosis of MEFs. MEFs prepared from individual embryos were treated with various dosages of AOB. Cell viability and apoptosis were detected by MTT and flow cytometry assays, respectively. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression. Functional annotation of differentially‑expressed genes was performed according to the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with the control group, ~50% of MEF cells were inhibited following treatment with a 400 μg/ml concentration of AOB. Treatment with 400 μg/ml AOB for 72 h significantly increased the apoptotic rate of MEF cells compared with the control group. Following treatment with AOB, dehydrogenase/reductase 9, phospholipase A2 group IVE and platelet derived growth factor B were downregulated, while 17 other genes were upregulated in MEF cells. Treatment with AOB markedly increased the expression of phosphorylated extracellular signal‑regulated kinase (ERK), β‑catenin, transcription factor SOX‑17, calcium‑binding tyrosine phosphorylation‑regulated protein, and cholesterol side chain cleavage enzyme mitochondrial (P<0.01). Additionally, the ERK pathway inhibitor U0126 and Wnt pathway inhibitor dickkopf‑related protein 1 markedly suppressed the expression of the above genes (P<0.01). AOB may impact the expression of proteins associated with embryonic fibroblast reproduction and embryonic development through activation of the ERK and Wnt signaling pathways, thus influencing cellular processes. [ABSTRACT FROM AUTHOR]

The article informs that the researchers made artificial mouse embryos from stem cells, without sperm or eggs, and used an innovative bioreactor to nurture their creations for longer than any previous embryo models. It further discusses early development with embryo mimics made from an assortment of mouse or human stem cells; views of stem cell biologist Niels Geijsen of the Leiden University Medical Center, on issue; and also mentions about human induced pluripotent stem cells.

In vitro maturation (IVM) of oocytes has been a highly successful method for avoiding the occurrence of severe ovarian hyperstimulation syndrome in some patients during in vitro fertilization. However, the safety of the protocol, especially the long-term effects, is still an issue of debate. The current study is to investigate the long-term effects of IVM on mice through two generations and reveal its inter-generational effects as well. The data indicate that the rates of embryo resorption and fetal death in the F1 generation were significantly increased while the newborn survival rate in the F1 and F2 generations were significantly decreased in the IVM group. Increased body weights in the F1 generation and mouse number per litter in the F2 generation were observed in both the IVM and VVM groups; however, no insulin resistance was detected. No significant differences were detected in birth defects, organ weights, testis histology and sperm motility, estrous cycle, and cognition among the IVM, VVM and N mice in either the F1 or F2 generations. Our results suggest that mouse IVM can affect pregnancy outcomes throughout two generations. IVM does not appear to influence the development and cognition of the offspring throughout two generations. [ABSTRACT FROM AUTHOR]

Our objective was to optimize the CA technique on mammal embryos. Materials and methods 1000 frozen 2-cell embryos from B6CBA mice were used. Based on a literature review, and after checking post-thaw embryo viability, the main outcome measures included: 1) comparison of the embryo recovery rate between 2 CA protocols (2 agarose layers and 3 agarose layers); 2) comparison of DNA damage by the CA on embryos with (ZP +) and without (ZP −) zona pellucida; and 3) comparison of DNA damage in embryos exposed to 2 genotoxic agents (H 2 O 2 and simulated sunlight irradiation (SSI)). DNA damage was quantified by the % tail DNA. Results 1) The recovery rate was 3,3% ( n = 5/150) with the 2 agarose layers protocol and 71,3% ( n = 266/371) with the 3 agarose layers protocol. 2) DNA damage did not differ statistically significantly between ZP − and ZP + embryos (12.60 ± 2.53% Tail DNA vs 11.04 ± 1.50 ( p = 0.583) for the control group and 49.23 ± 4.16 vs 41.13 ± 4.31 ( p = 0.182) for the H 2 O 2 group); 3) H 2 O 2 and SSI induced a statistically significant increase in DNA damage compared with the control group (41.13 ± 4.31% Tail DNA, 36.33 ± 3.02 and 11.04 ± 1.50 ( p < 0.0001)). The CA on mammal embryos was optimized by using thawed embryos, by avoiding ZP removal and by the adjunction of a third agarose layer. [ABSTRACT FROM AUTHOR]

Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage-matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high-resolution episcopic microscopy ( HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21-S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders ( DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis. [ABSTRACT FROM AUTHOR]

Histone H3 lysine 9 (H3K9) methylation is a hallmark of heterochromatin. H3K9 demethylation is crucial in mouse sex determination; The H3K9 demethylase Jmjd1a deficiency leads to increased H3K9 methylation at the Sry locus in embryonic gonads, thereby compromising Sry expression and causing male-to-female sex reversal. We hypothesized that the H3K9 methylation level at the Sry locus is finely tuned by the balance in activities between the H3K9 demethylase Jmjd1a and an unidentified H3K9 methyltransferase to ensure correct Sry expression. Here we identified the GLP/G9a H3K9 methyltransferase complex as the enzyme catalyzing H3K9 methylation at the Sry locus. Based on this finding, we tried to rescue the sex-reversal phenotype of Jmjd1a-deficient mice by modulating GLP/G9a complex activity. A heterozygous GLP mutation rescued the sex-reversal phenotype of Jmjd1a-deficient mice by restoring Sry expression. The administration of a chemical inhibitor of GLP/G9a enzyme into Jmjd1a-deficient embryos also successfully rescued sex reversal. Our study not only reveals the molecular mechanism underlying the tuning of Sry expression but also provides proof on the principle of therapeutic strategies based on the pharmacological modulation of epigenetic balance. [ABSTRACT FROM AUTHOR]

Caudal somites are generated from a pool of progenitor cells located in the tailbud region. These progenitor cells form the presomitic mesoderm that gradually differentiates into somites under the action of the segmentation clock. The signals responsible for tailbud mesoderm progenitor pool maintenance during axial elongation are still elusive. Here, we show that Bmp signaling is sufficient to activate the entire mesoderm progenitor gene signature in primary cultures of caudal mesoderm cells. Bmp signaling acts through the key regulatory genes brachyury (T) and Nkx1-2 and contributes to the activation of several other regulators of the mesoderm progenitor gene network. In the absence of Bmp signaling, tailbud mesoderm progenitor cells acquire aberrant gene expression signatures of the heart, blood, muscle and skeletal embryonic lineages. Treatment of embryos with the Bmp inhibitor noggin confirmed the requirement for Bmp signaling for normal T expression and the prevention of abnormal lineage marker activation. Together, these results identify Bmp signaling as a non-cell-autonomous signal necessary for mesoderm progenitor cell homeostasis. [ABSTRACT FROM AUTHOR]

ABSTRACT Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 μg mL−1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25-100 μg mL−1 ginsenoside Rb1 was associated with increased resorption of post-implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mg kg−1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses-triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre- and post-implantation development of mouse embryos, both in vitro and in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990-2003, 2017. [ABSTRACT FROM AUTHOR]

Background: In human IVF, embryos cultured with a lower O2 tension (5%) can give rise to higher success rates when compared with normoxic conditions (20%). However, the mechanisms behind the beneficial effects of reduced oxygen tension in embryogenesis remain unclear. The aim of this study was to evaluate the expression of oxygen related and antioxidant genes and mitochondrial function in mouse embryo cultured under hypoxic and normoxic conditions, to investigate the beneficial effect of low oxygen tension in preimplantation embryogenesis. Methods: Two-cell ICR mouse embryos were cultured to blastocysts under different oxygen tension (3% and 20%). The gene expression of oxygen-related proteins (hypoxia-inducible factor, HIF), HIF targets (vascular endothelial growth factor, VEGF; glucose transporter 3, GLUT-3) and antioxidants (manganese superoxide dismutase, MnSOD; peroxiredoxin 5, PRDX5) were assessed using quantitative RT-PCR and implantation-related protein (Leukemia Inhibitory Factor Receptor, LIFR) was validated by immunofluorescence. Apoptosis, mitochondrial membrane potential (MtMP) and ROS levels were measured by TUNEL, JC-1 and DCFDA assays, respectively. Results: Blastocyst development rate (92.3% vs. 79.4%) and hatch rate (80% vs. 70.4%) were both higher in embryos cultured in 3% O2 than in 20% O2. The transcription levels of MnSOD, PRDX5, VEGF and GLUT-3 also significantly increased in 3% O2 compared with 20% O2 (P < 0.05). Immunofluorescence showed that the intensity of staining for HIF-2α, MnSOD and LIFR were higher in 3% O2. Blastocysts cultured under 3% O2 exhibited significantly higher MtMP compared with 20% O2. ROS and Apoptosis levels were significantly higher in the 20% O2 group than in the 3% O2 group (P <0.05). Conclusions: Low O2 tension may improve embryo viability by increasing expression of antioxidant enzymes and glucose transporter activities. It provides an environment conducive to viability by upregulation of LIFR/VEGF and increased MtMP which could enhance implantation potential and reduce apoptosis in mouse blastocyst. These effects may be initiated and regulated by HIF-2α,akeymediatorinahypoxicenvironment. [ABSTRACT FROM AUTHOR]

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injury. [ABSTRACT FROM AUTHOR]

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. [ABSTRACT FROM AUTHOR]

The separation of embryonic from extra-embryonic tissues within the inner cell mass to generate the epiblast (EPI), which will form the new organism, from the primitive endoderm (PE), which will form the yolk sac, is a crucial developmental decision. Here,we identify a chromatin modifier, Satb1, with a distinct role in this decision. Satb1 is differentially expressed within 16-cell-stage embryos, with higher expression levels in the inner cell mass progenitor cells. Depleting Satb1 increases the number of EPI cells at the expense of PE. This phenotype can be rescued by simultaneous depletion of both Satb1 and Satb2, owing to their antagonistic effect on the pluripotency regulator Nanog. Consequently, increasing Satb1 expression leads to differentiation into PE and a decrease in EPI, as a result of the modulation of expression of several pluripotency- and differentiationrelated genes by Satb1. Finally, we show that Satb1 is a downstream target of the Fgf signalling pathway, linking chromatin modification and Fgf signalling. Together, these results identify a role for Satb1 in the lineage choice between pluripotency and differentiation and further our understanding of early embryonic lineage segregation. [ABSTRACT FROM AUTHOR]

Purpose: Successful in vitro fertilization (IVF) relies on sound laboratory methods and culture conditions which depend on sensitive quality control (QC) testing. This study aimed to improve the sensitivity of mouse embryo assays (MEA) for detection of mineral oil toxicity. Methods: Five experiments were conducted to study modifications of the standard mouse embryo assay (MEA) in order to improve sensitivity using clinical grade mineral oil with known peroxide concentrations. Assessment of blastocyst development at either 96 h or in an extended MEA (eMEA) to 144 h was tested in each experiment. In experiment 1, ability to detect peroxides in oil was compared in the MEA, eMEA, and cell number at 96 h. In experiment 2, serial dilutions of peroxide in oil were used along with time-lapse imaging to compare sensitivity of the morphokinetic MEA to the eMEA. Culture conditions that may affect assay sensitivity were assessed in experiments 3-5, which examined the effect of group versus individual culture, oxygen concentration, and protein supplementation. Results: Extended MEA and cell counts identified toxicity not detected by the routine endpoint of blastocyst rate at 96 h. The eMEA was fourfold more sensitive than the standard MEA, and this sensitivity was similar to the morphokinetic MEA. Group culture had a protective effect against toxicity, while oxygen concentration did not affect blastocyst development. Protein supplementation with HSA had a protective effect on blastocyst development in eMEA. Conclusions: The standard MEA used by manufacturers does not detect potentially lethal toxicity of peroxides in mineral oil. While group culture may mask toxicity, protein supplementation and oxygen concentration have minimal effect on assay sensitivity. The eMEA and time-lapse morphokinetic assessment are equally effective in detection of peroxide toxicity and thus provide manufacturers and end-users a simple process modification that can be readily adopted into an existing QC program. [ABSTRACT FROM AUTHOR]

The group C SOX transcription factors SOX4, −11 and −12 play important and mutually overlapping roles in development of a number of organs. Here, we examined the role of SoxC genes during gonadal development in mice. All three genes were expressed in developing gonads of both sexes, predominantly in somatic cells, with Sox4 being most strongly expressed. Sox4 deficiency resulted in elongation of both ovaries and testes, and an increased number of testis cords. While female germ cells entered meiosis normally, male germ cells showed reduced levels of differentiation markers Nanos2 and Dnmt3l and increased levels of pluripotency genes Cripto and Nanog , suggesting that SOX4 may normally act to restrict the pluripotency period of male germ cells and ensure their proper differentiation. Finally, our data reveal that SOX4 (and, to a lesser extent, SOX11 and −12) repressed transcription of the sex-determining gene Sox9 via an upstream testis-specific enhancer core (TESCO) element in fetal gonads, raising the possibility that SOXC proteins may function as transcriptional repressors in a context-dependent manner. [ABSTRACT FROM AUTHOR]

In vitro antioxidant virtue and life-prolonging effect of phycoerythrin (PE; a pigment protein isolated from Phormidium sp. A09DM) have been revealed in our previous reports (Sonani et al. in Age 36:9717, 2014a; Sonani et al. in Process Biochem 49:1757-1766, 2014b). It has been hypothesized that the PE expands life span of Caenorhabditis elegans (bears large resemblance with human aging pathways) due to its antioxidant virtue. This hypothesis is tested in present study by checking the effect of PE on intracellular reactive oxygen species (ROS) generation and associated physiological deformities using mouse and human skin fibroblasts, C. elegans, and Drosophila melanogaster Oregon R and by divulging PE's structural attributes responsible for its antioxidant asset. PE treatment displayed noteworthy decrease of 67, 48, and 77 % in ROS level in mouse fibroblast ( 3T3-L1), human fibroblast, and C. elegans N2, respectively, arisen under chemical-induced oxidative stress. PE treatment delayed the development of paraquat-induced Alzheimer phenotype by 14.5 % in C. elegans CL4176. Furthermore, PE improved the locomotion of D. melanogaster Oregon R under oxidative stress with simultaneous up-regulation in super-oxide dismutase and catalase activities. The existence of 52 Glu + Asp + His + Thr residues (having metal ion sequestration capacity), 5 phycoerythrobilin chromophores (potential electron exchangers) in PE's primary structure, and significant hydrophobic patches on the surface of its α- and β-subunits are supposed to collectively contribute in the antioxidant virtues of PE. Altogether, results support the hypothesis that it is the PE's antioxidant asset, which is responsible for its life-prolonging effect and thus could be exploited in the therapeutics of ROS-associated abnormalities including aging and neurodegeneration in eukaryotes. [ABSTRACT FROM AUTHOR]

Early embryos of all mammalian species contain morphologically distinct but transcriptionally silent nucleoli called the nucleolar precursor bodies (NPBs), which, unlike normal nucleoli, have been poorly studied at the biochemical level. To bridge this gap, here we examined the occurrence of RNA and proteins in early mouse embryos with two fluorochromes - an RNA-binding dye pyronin Y (PY) and the protein-binding dye fluorescein-50-isothiocyanate (FITC). The staining patterns of zygotic NPBs were then compared with those of nucleolus-like bodies (NLBs) in fully grown surrounded nucleolus (SN)-type oocytes, which are morphologically similar to NPBs. We show that both entities contain proteins, but unlike NLBs, NPBs are significantly impoverished for RNA. Detectable amounts of RNA appear on the NPB surface only after resumption of rDNA transcription and includes pre-rRNAs and 28S rRNA as evidenced by fluorescence in situ hybridisation with specific oligonucleotide probes. Immunocytochemical assays demonstrate that zygotic NPBs contain rRNA processing factors fibrillarin, nucleophosmin and nucleolin, while UBF (the RNA polymerase I transcription factor) and ribosomal proteins RPL26 and RPS10 are not detectable. Based on the results obtained and data in the contemporary literature, we suggest a scheme of NPB assembly and maturation to normal nucleoli that assumes utilisation of maternally derived nucleolar proteins but of nascent rRNAs. [ABSTRACT FROM AUTHOR]

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with aberrant epigenetic nuclear reprogramming. It has been demonstrated that treatment with histone deacetylase inhibitors (HDACis) enhances developmental potential of SCNT embryos. Previous studies in many species revealed that treatment of SCNT embryos with trichostatin A (TSA)-an HDACi-significantly enhances the in vitro development of SCNT embryos. In this study, we compared two different SCNT protocols with TSA and investigate, for the first time, the effect of another new HDACi, PXD101 (belinostat), on in vitro development of mouse SCNT embryo. Rates of blastocyst development in mouse SCNT embryos treated with either 5 nM TSA during (6 hours) and after (4 hours) activation (39.1%) or with 50 nM PXD101 during (6 hours) and after (4 hours) activation (40.2%) were significantly higher than those of nontreated SCNT embryos (11.5%) and both treatments also significantly improved the subsequent establishment of NT-ESCs in comparison with the nontreated group (38.1% and 40.9% vs. 11.8%). In conclusion, we optimized the TSA concentration and treatment timing and, for the first time, investigated the effect of PXD101 on mouse development of SCNT embryos and establishment of NT-ESCs. [ABSTRACT FROM AUTHOR]

During preimplantation development, mouse embryos form two types of cells, the trophoectoderm ( TE) and inner cell mass ( ICM), by the early blastocyst stage. This process does not require maternal factors localized in the zygotes, and embryos self-organize at the blastocyst stage through intercellular communications. In terms of the mechanisms of cell fate specification, three historical models have been proposed: the positional model, and the original and newer versions of the polarity model. Recent studies have revealed that the intercellular Hippo signaling pathway plays a central role in the specification of the first cell fates. Hippo signaling is active in the inner cells but inactive in the outer cells. The Hippo-active inner and Hippo-inactive outer cells take the fates of the ICM and the TE, respectively. At the 32-cell stage, E-cadherin-mediated cell-cell adhesion and cell polarization by the Par- aPKC system activates and inactivates the Hippo pathway, respectively. Both mechanisms involve regulation of angiomotin, and cooperation of these mechanisms establishes cell position-dependent activation of Hippo signaling. At the 16-cell stage, however, asymmetric cell division produces the initial differences in Hippo signaling. At this stage, cell polarity is controlled by both Par- aPKC-dependent and -independent mechanisms. All three historical models are explained by the different regulations and roles of Hippo signaling. Based on these findings, I would like to propose the model by which the differences in Hippo signaling among blastomeres is first produced by asymmetric cell division and then enhanced and stabilized by cell position-dependent mechanisms until their fates are fixed. [ABSTRACT FROM AUTHOR]

Osteogenesis and angiogenesis play the prominent role in the bone regeneration. In this study, deferoxamine (DFO), an induced agent for osteogenesis and angiogenesis, was modified onto the surface of poly( D,L -lactide) (PDLLA) membrane via a facile and convenient approach based on the self-polymerization of dopamine (DOPA). The surface composition, morphology, hydrophilicity and surface energy of the original and modified PDLLA membranes were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), scanning electronic microscopy (SEM), atomic force microscopy (AFM) and contact angle measurement. The surface roughness and hydrophilicity of the PDLLA membrane were obviously increased by introducing either the single polydopamine (PDOPA) or the dual layers of PDOPA and DFO. In vitro cells culture experiments indicated that both the PDLLA/PDOPA and PDLLA/PDOPA-DFO composite membranes were more beneficial to the attachment, proliferation and spreading of MC3T3-E1 cells and HUVECs compared to the original PDLLA membrane. The PDLLA/PDOPA-DFO membrane was supportive for the proliferation of both MC3T3-E1 cells and HUVECs, and especially for HUVECs. The results suggested that the as-prepared PDLLA/PDOPA-DFO composite can be expected to be used as a promising bone regenerative material with promoted angiogenesis. [ABSTRACT FROM AUTHOR]