Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

International Nuclear Information System (INIS)

Belkin, V.M.; Volodarskaya, S.M.

1986-01-01

The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

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Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

2017-12-13

Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos.

Science.gov (United States)

Liu, Haijun; Peng, Hui; Liu, Fang; Ma, Qun; Zhang, Wenchang

2016-05-01

The present study aimed to detect the expression of β-galactosidase during long-term cultured goat skin fibroblasts and investigate the effects of donor goat age, sex, and cell passage on senescence and the effects of donor cell passage on in vitro development of nuclear transfer embryos. The results showed that, in the same cell passage, more β-galactosidase-positive cells were detected in cells from older donors than younger donors. Irrespective of the donor age, the number of positive cells was higher in later passages from passages 20 to 50. In the same passage from 20 to 50, the β-galactosidase-positive rate was higher in cells from 5-yr female goat than 5-yr male goat. Using fibroblasts from male goats at various passages as donor cells, reconstructed embryos had similar fusion and cleavage rates, but the blastocyst rate was higher for cells at passages 10 and 20 than passage 30. In conclusion, donor goat age and cell passage had significant effects on the β-galactosidase-positive rate; also, cells from 5-yr female goat had a higher β-galactosidase-positive rate than those from 5-yr male goat, and the donor cell passage affected the developmental potential of nuclear transfer embryos.

Efficiency of two enucleation methods connected to handmade cloning to produce transgenic porcine embryos

DEFF Research Database (Denmark)

Li, J; Villemoes, K; Zhang, Y

2009-01-01

The purpose of our work was to establish an efficient-oriented enucleation method to produce transgenic embryos with handmade cloning (HMC). After 41â€"42 h oocytes maturation, the oocytes were further cultured with or without 0.4 μg/ml demecolcine for 45 min [chemically assisted handmade...... cytoplasts without extrusion cones or PB were selected as recipients. Two cytoplasts were electrofused with one transgenic fibroblasts expressing green fluorescent protein (GFP), while non-transgenic fibroblasts were used as controls. Reconstructed embryos were cultured in Well of Wells (WOWs) with porcine......%) of cloned embryos with GFP transgenic fibroblast cells after CAHE vs OHE. With adjusted time-lapse for zonae-free cloned embryos cultured in WOWs with PZM-3, it was obvious that in vitro developmental competence after CAHE was compromised when compared with the OHE method. OHE enucleation method seems...

Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts.

Science.gov (United States)

Pistocchi, A; Fazio, G; Cereda, A; Ferrari, L; Bettini, L R; Messina, G; Cotelli, F; Biondi, A; Selicorni, A; Massa, V

2013-10-17

Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the 'cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

Different Donor Cell Culture Methods Can Influence the Developmental Ability of Cloned Sheep Embryos.

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LiBing Ma

Full Text Available It was proposed that arresting nuclear donor cells in G0/G1 phase facilitates the development of embryos that are derived from somatic cell nuclear transfer (SCNT. Full confluency or serum starvation is commonly used to arrest in vitro cultured somatic cells in G0/G1 phase. However, it is controversial as to whether these two methods have the same efficiency in arresting somatic cells in G0/G1 phase. Moreover, it is unclear whether the cloned embryos have comparable developmental ability after somatic cells are subjected to one of these methods and then used as nuclear donors in SCNT. In the present study, in vitro cultured sheep skin fibroblasts were divided into four groups: (1 cultured to 70-80% confluency (control group, (2 cultured to full confluency, (3 starved in low serum medium for 4 d, or (4 cultured to full confluency and then further starved for 4 d. Flow cytometry was used to assay the percentage of fibroblasts in G0/G1 phase, and cell counting was used to assay the viability of the fibroblasts. Then, real-time reverse transcription PCR was used to determine the levels of expression of several cell cycle-related genes. Subsequently, the four groups of fibroblasts were separately used as nuclear donors in SCNT, and the developmental ability and the quality of the cloned embryos were compared. The results showed that the percentage of fibroblasts in G0/G1 phase, the viability of fibroblasts, and the expression levels of cell cycle-related genes was different among the four groups of fibroblasts. Moreover, the quality of the cloned embryos was comparable after these four groups of fibroblasts were separately used as nuclear donors in SCNT. However, cloned embryos derived from fibroblasts that were cultured to full confluency combined with serum starvation had the highest developmental ability. The results of the present study indicate that there are synergistic effects of full confluency and serum starvation on arresting fibroblasts in

Interleukin-1β attenuates myofibroblast formation and extracellular matrix production in dermal and lung fibroblasts exposed to transforming growth factor-β1.

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Masum M Mia

Full Text Available One of the most potent pro-fibrotic cytokines is transforming growth factor (TGFβ. TGFβ is involved in the activation of fibroblasts into myofibroblasts, resulting in the hallmark of fibrosis: the pathological accumulation of collagen. Interleukin-1β (IL1β can influence the severity of fibrosis, however much less is known about the direct effects on fibroblasts. Using lung and dermal fibroblasts, we have investigated the effects of IL1β, TGFβ1, and IL1β in combination with TGFβ1 on myofibroblast formation, collagen synthesis and collagen modification (including prolyl hydroxylase, lysyl hydroxylase and lysyl oxidase, and matrix metalloproteinases (MMPs. We found that IL1β alone has no obvious pro-fibrotic effect on fibroblasts. However, IL1β is able to inhibit the TGFβ1-induced myofibroblast formation as well as collagen synthesis. Glioma-associated oncogene homolog 1 (GLI1, the Hedgehog transcription factor that is involved in the transformation of fibroblasts into myofibroblasts is upregulated by TGFβ1. The addition of IL1β reduced the expression of GLI1 and thereby also indirectly inhibits myofibroblast formation. Other potentially anti-fibrotic effects of IL1β that were observed are the increased levels of MMP1, -2, -9 and -14 produced by fibroblasts exposed to TGFβ1/IL1β in comparison with fibroblasts exposed to TGFβ1 alone. In addition, IL1β decreased the TGFβ1-induced upregulation of lysyl oxidase, an enzyme involved in collagen cross-linking. Furthermore, we found that lung and dermal fibroblasts do not always behave identically towards IL1β. Suppression of COL1A1 by IL1β in the presence of TGFβ1 is more pronounced in lung fibroblasts compared to dermal fibroblasts, whereas a higher upregulation of MMP1 is seen in dermal fibroblasts. The role of IL1β in fibrosis should be reconsidered, and the differences in phenotypical properties of fibroblasts derived from different organs should be taken into account in future

Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

Science.gov (United States)

Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

2017-01-01

The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

Gene expression profiling in zebrafish embryos exposed to diclofenac, an environmental toxicant.

Science.gov (United States)

De Felice, Bruna; Copia, Luisa; Guida, Marco

2012-03-01

Pharmaceuticals are continually released in the environment and therefore pollution from drugs is a pressing problem in the environment. Diclofenac, 2-[(2,6-dichlorophenyl)amino]phenylacetic acid is a FDA approved non-steroidal anti-inflammatory drug (NSAID) for the treatment of inflammation. This pharmaceutical has been found as pollutant in superficial waters. Danio rerio (zebrafish) embryo has been used as a model organism for acute pollutant toxicity tests in order to identify morphological alterations in development and death rate. Through the combination of mRNA differential display and quantitative Real Time experiments, we analyzed the alterations of gene expression in zebrafish embryos left to develop in the presence of diclofenac and thereby assess the molecular mechanism involved in ecotoxicity of diclofenac polluted waters. This approach, in embryos exposed to 1.25 mg/l drug for 48 h, allowed identifying 36 different genes, with both known and unknown functions, whose transcription is differentially regulated. The identity and ontological classification of these genes is presented. The wide variety of functional classes of transcripts isolated in this screen reflects the diverse spectrum of influences operating across diclofenac exposure. Of these 36 genes, several have been selected for detailed quantitative Real Time analysis to validate the screen. Our results, for the first time, provide an insight into some of the varied and novel molecular networks following zebrafish exposure to diclofenac polluted waters.

The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio Embryos and Eleutheroembryos Exposed to Methylmercury.

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Francisco X Mora-Zamorano

Full Text Available This study is an adaptation of the nicotine-evoked locomotor response (NLR assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf, however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf by means of acute nicotine exposure (30-240μM. Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM. Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine.

Radiosensitive target in the early mouse embryo exposed to very low doses of ionizing radiation

International Nuclear Information System (INIS)

Wiley, Lynn M.; Raabe, Otto G.; Khan, Rakhshi; Straume, Tore

1994-01-01

We exposed mouse preimplantation embryos in vitro to either tritiated water (HTO) or tritiated thymidine (TdR) to determine whether the radiosensitive target was nuclear or extranuclear for embryonic cell proliferation disadvantage in the mouse embryo chimera assay. 8-cell embryos were incubated in either HTO or TdR for 2 h and paired with non-irradiated control embryos to form chimeras. Chimeras were cultured for an average of 20.2 h to allow for 2-3 cell cycles and then partially dissociated to obtain the number of progeny cells contributed by the two partner embryos for each chimera. These values were expressed as a 'proliferation ratio' (number of cells from the irradiated embryo: total number of cells in the chimera). A ratio significantly less than 0.50 indicates that the experimental embryo expressed an embryonic cell proliferation disadvantage, which is the endpoint of this assay. The activity concentrations of HTO and TdR were adjusted so that both would deliver comparable mean absorbed nuclear doses during the combined initial 2-h irradiation incubation and subsequent 20.2 h chimera incubation periods. Although nuclear doses were comparable under these conditions, the extranuclear dose delivered by the uniformly distributed HTO was about 100 times greater than the extranuclear dose delivered by TdR for each given nuclear dose. Consequently, obtaining mean TdR proliferation ratios≤mean HTO proliferation ratios would be evidence for a nuclear target while obtaining mean HTO proliferation ratios< mean TdR proliferation ratios would be evidence for an extranuclear target. TdR consistently produced lower mean proliferation ratios over a range of doses from 0.14 Gy to 0.43 Gy. Therefore, we conclude that the radiosensitive target for this endpoint is nuclear

DNA strand breaks detected in embryos of the adult snails, Potamopyrgus antipodarum, and in neonates exposed to genotoxic chemicals

International Nuclear Information System (INIS)

Vincent-Hubert, Françoise; Revel, Messika; Garric, Jeanne

2012-01-01

We tested the freshwater mudsnail Potamopyrgus antipodarum, which is a species that has already been used for endocrine-disrupting compounds (EDCs) to determine whether early life stages of aquatic organisms are sensitive to genotoxic chemicals. For this purpose, we first developed the alkaline comet assay on adults, embryos, and neonates. The comet assay protocol was validated on both embryonic cells exposed in vitro to hydrogen peroxide and adult snails in the reproducing stage exposed to methyl methane sulfonate. During the latter experiment, DNA strand breaks were investigated on both embryonic cells and on adult gill cells. The second part of this study investigated the stability of DNA strand breaks in adult reproducing snails and neonates exposed to cadmium (Cd) and bisphenol A for 8 days. Hydrogen peroxide-induced DNA strand breaks in vitro in isolated embryonic cells. Exposure of adult reproducing snails to methyl methane sulfonate for 24 h induced DNA strand breaks in embryos. Bisphenol A induced a significant increase in the DNA strand-break level in whole embryonic cells and whole neonate cells. Cd was genotoxic for both embryos and neonates during the exposure time and also after 7 days of depuration, suggesting that Cd could inhibit DNA repair enzymes. These preliminary results on this original model have encouraged us to consider the impact of genotoxic environmental contaminants on the F1 generation.

DNA strand breaks detected in embryos of the adult snails, Potamopyrgus antipodarum, and in neonates exposed to genotoxic chemicals

Energy Technology Data Exchange (ETDEWEB)

Vincent-Hubert, Francoise, E-mail: francoise.vincent-hubert@irstea.fr [Unite de Recherche Hydrosystemes et Bioprocedes, equipe BELCA, IRSTEA/CEMAGREF, 1 rue Pierre-Gilles de Gennes, CS10030, 92761 Antony cedex, 92163 Antony (France); Revel, Messika [Unite de Recherche Hydrosystemes et Bioprocedes, equipe BELCA, IRSTEA/CEMAGREF, 1 rue Pierre-Gilles de Gennes, CS10030, 92761 Antony cedex, 92163 Antony (France); Garric, Jeanne [MALY Laboratoire d' ecotoxicologie, IRSTEA/CEMAGREF, 23 bis Quai Chauveau, 69006 Lyon (France)

2012-10-15

We tested the freshwater mudsnail Potamopyrgus antipodarum, which is a species that has already been used for endocrine-disrupting compounds (EDCs) to determine whether early life stages of aquatic organisms are sensitive to genotoxic chemicals. For this purpose, we first developed the alkaline comet assay on adults, embryos, and neonates. The comet assay protocol was validated on both embryonic cells exposed in vitro to hydrogen peroxide and adult snails in the reproducing stage exposed to methyl methane sulfonate. During the latter experiment, DNA strand breaks were investigated on both embryonic cells and on adult gill cells. The second part of this study investigated the stability of DNA strand breaks in adult reproducing snails and neonates exposed to cadmium (Cd) and bisphenol A for 8 days. Hydrogen peroxide-induced DNA strand breaks in vitro in isolated embryonic cells. Exposure of adult reproducing snails to methyl methane sulfonate for 24 h induced DNA strand breaks in embryos. Bisphenol A induced a significant increase in the DNA strand-break level in whole embryonic cells and whole neonate cells. Cd was genotoxic for both embryos and neonates during the exposure time and also after 7 days of depuration, suggesting that Cd could inhibit DNA repair enzymes. These preliminary results on this original model have encouraged us to consider the impact of genotoxic environmental contaminants on the F1 generation.

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

Science.gov (United States)

Di Renzo, Francesca; Rossi, Federica; Bacchetta, Renato; Prati, Mariangela; Giavini, Erminio; Menegola, Elena

2011-06-01

The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology. © 2011 Wiley-Liss, Inc.

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation.

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Wang, Yuanyuan; Chen, Chen; Deng, Ziyu; Bian, Erbao; Huang, Cheng; Lei, Ting; Lv, Xiongwen; Liu, Liping; Li, Jun

2017-03-01

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately. Copyright © 2016. Published by Elsevier B.V.

AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

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Jaguin, Marie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes Cedex (France); Lecureur, Valérie, E-mail: valerie.lecureur@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France)

2015-06-15

Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantly increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants. - Highlights: • PDGF-B expression and secretion are increased by DEPe exposure in human M1 and M2 MΦ. • DEPe-induced PDGF-B expression is aryl-hydrocarbon-dependent. • DEPe-exposed M1 MΦ secrete sufficient PDGF-B to increase lung fibroblast proliferation.

Embryo developmental capacity of oocytes fertilised by sperm of mouse exposed to forced swimming stress

International Nuclear Information System (INIS)

Ghasem, S.; Majid, J.; Shiva, R.

2013-01-01

Objective: To assess developmental capacity of fertilised oocytes by sperm of mouse exposed to forced swimming stress. Methods: The experimental study was conducted at the Physiology Research Center of Ahvaz Jundishapur University of Medical Sciences, from August 2011 to January 2012. It comprised 20 adult male and 10 female mice. The male mice were randomly divided into two equal groups (n=10): control and experimental. Animals of the experimental group were submitted to forced swimming stress. All male mice were euthanised and the cauda epididymis removed before contents were squeezed out. A pre-incubated capacitated sperm was gently added to the freshly collected ova of the two groups of study. The combined sperm-oocyte suspension was incubated for 4-6 hours under a condition of 5% Carbon dioxide and 37 degree C temperature. The ova were then washed through several changes of medium and finally incubated. Fertilisation was assessed by recording the number of 1-cell embryos 4-6 hours after insemination. The 1-cell embryos were allowed to further develop in vitro for about 120 hours. Development of embryos everyday and during 5 days of culture was observed by using inverted microscope. SPSS 13.0.1 was used for statistical analysis. Results: The percentage of oocytes fertilised was 75:96 (78.12+-4.8%) and 50:10 (49.5+-3.9%) in the control and experimental groups, respectively. The difference was significant (p 0.05) between the two groups in terms of speed and developmental capacity of blastocysts. Conclusions: Fertilisation capacity of male mice affected by forced swimming stress and also the developmental capacity of oocyte fertilised by sperm of mouse exposed to forced swimming stress decreased. (author)

Embryo developmental capacity of oocytes fertilised by sperm of mouse exposed to forced swimming stress.

Science.gov (United States)

Ghasem, Saki; Majid, Jasemi; Shiva, Razi

2013-07-01

To assess developmental capacity of fertilised oocytes by sperm of mouse exposed to forced swimming stress. The experimental study was conducted at the Physiology Research Center of Ahvaz Jundishapur University of Medical Sciences, from August 2011 to January 2012. It comprised 20 adult male and 10 female mice. The male mice were randomly divided into two equal groups (n=10): control and experimental. Animals of the experimental group were submitted to forced swimming stress. All male mice were euthanised and the cauda epididymis removed before contents were squeezed out. A pre-incubated capacitated sperm was gently added to the freshly collected ova of the two groups of study. The combined sperm-oocyte suspension was incubated for 4-6 hours under a condition of 5% Carbon dioxide and 37 degreeC temperature. The ova were then washed through several changes of medium and finally incubated. Fertilisation was assessed by recording the number of 1-cell embryos 4-6 hours after insemination. The 1-cell embryos were allowed to further develop in vitro for about 120 hours. Development of embryos everyday and during 5 days of culture was observed by using inverted microscope. SPSS 13.0.1 was used for statistical analysis. The percentage of oocytes fertilised was 75:96 (78.12+/-4.8%) and 50:10 (49.5+/-3.9%) in the control and experimental groups, respectively. The difference was significant (p 0.05) between the two groups in terms of speed and developmental capacity of blastocysts. Fertilisation capacity of male mice affected by forced swimming stress and also the developmental capacity of oocyte fertilised by sperm of mouse exposed to forced swimming stress decreased.

Influences of somatic donor cell sex on and embryo development following somatic cell nuclear transfer in pigs

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Jae-Gyu Yoo

2017-04-01

Full Text Available Objective The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. Methods Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. Results The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8 was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT groups (31.4±8.3 to 33.4±11.1. After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05 between sexes. Conclusion The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Review of NCRP radiation dose limit for embryo and fetus in occupationally-exposed women

International Nuclear Information System (INIS)

Anon.

1977-01-01

On the basis of the current review, the NCRP has decided to make no change in the current recommendation of its radiation dose limit to the unborn. The NCRP recommendation is restated here as follows: During the entire gestation period, the maximum permissible dose equivalent to the embryo-fetus from occupational exposure of the expectant mother should be 0.5 rem. Since the preparation of the 1971 report there has been no new evidence concerning teratogenic or carcinogenic effects of irradiation of the embryo-fetus that would justify a change in the limit in either direction. It is implicit in this position and recommendation that women who can reasonably be expected to be pregnant should not, in certain instances, be exposed to the same radiation environment as women who are not considered fertile or as men. This applies particularly to conditions where radiation workers can receive dose equivalents of 0.5 rem or more in short periods

Effects of recipient oocyte age and interval from fusion to activation on development of buffalo (Bubalus bubalis) nuclear transfer embryos derived from fetal fibroblasts.

Science.gov (United States)

Lu, F; Jiang, J; Li, N; Zhang, S; Sun, H; Luo, C; Wei, Y; Shi, D

2011-09-15

The objective was to investigate the effect of recipient oocyte age and the interval from activation to fusion on developmental competence of buffalo nuclear transfer (NT) embryos. Buffalo oocytes matured in vitro for 22 h were enucleated by micromanipulation under the spindle view system, and a fetal fibroblast (pretreated with 0.1 μg/mL aphidicolin for 24 h, followed by culture for 48 h in 0.5% fetal bovine serum) was introduced into the enucleated oocyte, followed by electrofusion. Both oocytes and NT embryos were activated by exposure to 5 μM ionomycin for 5 min, followed by culture in 2 mM 6-dimethyl-aminopurine for 3 h. When oocytes matured in vitro for 28, 29, 30, 31, or 32 h were activated, more oocytes matured in vitro for 30 h developed into blastocysts in comparison with oocytes matured in vitro for 32 h (31.3 vs 19.9%, P fusion (P fusion. However, 3 of 16 recipients were pregnant following transfer of blastocysts developed from the NT embryos activated at 3 h after fusion, and two of these recipients maintained pregnancy to term. We concluded that the developmental potential of buffalo NT embryos was related to recipient oocyte age and the interval from fusion to activation. Copyright © 2011 Elsevier Inc. All rights reserved.

Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells.

OpenAIRE

Miyasaka, M; Takami, Y; Inoue, H; Hakura, A

1991-01-01

The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed ph...

Correction of β-thalassemia mutant by base editor in human embryos

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Puping Liang

2017-09-01

Full Text Available Abstract β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.

Science.gov (United States)

Sylvain, Nicole J; Brewster, Daniel L; Ali, Declan W

2010-01-01

Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3 days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion. Copyright 2010 Elsevier Inc. All rights reserved.

Biosynthesis of collagen by fibroblasts kept in culture

International Nuclear Information System (INIS)

Machado-Santelli, G.M.

1978-01-01

The sinthesis of collagen is studied in fibroblasts of different origins with the purpose of obtaining an appropriate system for the study of its biosynthesis and processing. The percentage of collagen synthesis vary according to the fibroblast origin. Experiences are performed with fibroblasts kept in culture from: chicken - and guinea pig embryos, carragheenin - induced granulomas in adult guinea pig and from human skin. The collagen pattern synthesized after acetic acid - or saline extractions in the presence of inhibitors is also determined. This pattern is then assayed by poliacrilamide - 5% - SDS gel electrophoresis accompanied by fluorography. The importance of the cell culture system in the elucidation of collagen biosynthesis is pointed out. (M.A.) [pt

Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

Science.gov (United States)

Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

2016-09-02

Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Aquatic toxicity assessment of single-walled carbon nanotubes using zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Pan Huichin; Lin Yujun; Li Mengwei [Department of Biomedical Sciences, Chung Shan Medical University, Taichung 40201, Taiwan (China); Chuang Hanni; Chou Chengchung, E-mail: bioccc@ccu.edu.tw, E-mail: hp29@csmu.edu.tw [Department of Life Science, National Chung Cheng University, Min-Hsiung, 62102 Taiwan (China)

2011-07-06

Zebrafish embryos selected at the 64-cell stage were exposed to various concentrations of amide functionalized single-walled carbon nanotubes (SWCNTs) ranging from 1 to 10 {mu}g/ml dissolved in 1% Pluronic F-68 (a cell culture grade surfactant), and the development of embryos was examined from 24 to 120 hours post fertilization (hpf). Incubation of embryos in 1% F-68 did not induce overt abnormal phenotype as compared to the wild-type; neither did it cause significant mortality during the exposure period. Generally, there was a slight developmental delay in larvae treated with SWCNTs of 5 {mu}g/ml or above. Only larvae exposed to {>=} 5 {mu}g/ml SWCNTs showed significantly reduced survival rates. About 50% of the embryos exposed to 5 {mu}g/ml showed abnormal phenotypes at 24 hpf as compared to the control group. As development proceeds to 120 hpf, more embryos displayed defective morphology. A slight hatching delay was observed in embryos exposed to concentrations above 5 {mu}g/ml. There was a general reduction of body axes, including narrowed somite and shortened yolk stalk. In addition, pigmentation in the ventral trunk area was less than that observed in control group. The body lengths of the exposed embryos were decreased significantly at 48 hpf (3.11 mm in control vs. 3.00 mm in SWCNTs-exposed embryos). However, exposure to SWCNTs did not affect the number of somites. Other features that were noticed in the SWCNTs-exposed embryos included edema and shrinkage and blebbling of the epidermal lining. Most of these observed phenotypes persisted from 48 hpf through 120 hpf. Overall, the aforementioned results indicate that soluble amide-functionalized SWCNTs are toxic to zebrafish embryos at a minimum concentration of 5 {mu}g/ml.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos.

Science.gov (United States)

Li, Rong; Liu, Ying; Pedersen, Hanne Skovsgaard; Callesen, Henrik

2015-06-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time. In conclusion, exposure to ambient light can be harmful to embryo development, both when medium is exposed for a long period of time and, to a greater extent, when the embryo itself is exposed for >1 h. In practice, it is therefore recommended to protect both culture medium and porcine embryos against ambient light during in vitro handling in the laboratory.

Mental retardation occurring in embryo exposed in utero to the atomic bomb (Hiroshima)

International Nuclear Information System (INIS)

Ishikawa, Hiroya; Shimasaki, Akira; Fujiwara, Koichi; Harada, Masazumi; Minami, Ryuichi.

1978-01-01

This paper deals with a long term follow-up study on psychological symptoms in four patients with microscopically microcephaly induced by prenatal exposed to atomic bomb. They were exposed to atomic bomb at 8- and 12-week-embryos. The distance from the center of the explosion was 780 - 1180 m. All their mothers had acute radiation hazards. Their growths in the uterus were markedly damaged. Postnatal body weight ranged between 1,300 and 2,000 g. They were commonly characterized by microcephaly, physiological and mental retardation, stigmata of degeneracy, and skin symptoms, who were diagnosed as ''microcephaly induced by early prenatal exposure to atomic bomb'' by the research group for microcephaly in the Ministry of Health and Welfare. These common symptoms such as microcephaly, stigmata of degeneracy, and disturbed growth, but neurological symptoms such as motor aphasia were slighter in these patients than in those having congenital Minamata disease prenatal. These results suggested that the prognosis of these patients in whom 30 years have passed is unexpectedly serious. (Namekawa, K.)

Reduction in life span on normal human fibroblasts exposed to low-dose radiation in heavy-ion radiation field

International Nuclear Information System (INIS)

Suzuki, Masao; Yamaguchi, Chizuru; Yasuda, Hiroshi; Uchihori, Yukio; Fujitaka, Kazunobu

2003-01-01

We studied the effect of in vitro life span in normal human fibroblasts exposed to chronically low-dose radiation in heavy-ion radiation field. Cells were cultured in a CO 2 incubator, which was set in the irradiation room for biological study of heavy ions in the Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS), and exposed to scattered radiations produced with heavy-ion beams throughout the life span of the cell population. Absorbed dose, which was measured using a thermoluminescence dosimeter(TLD) and a Si-semiconductor detector, was to be 1.4 mGy per day when operating the HIMAC machine for biological experiments. The total population doubling number of the exposed cells reduced to 79-93% of non-exposed control cells in the three independent experiments. There is evidence that the exposure of chronically low-dose radiation in heavy-ion radiation field promotes the life-span reduction in cellular level. (author)

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium.

Science.gov (United States)

Chiarelli, Roberto; Martino, Chiara; Agnello, Maria; Bosco, Liana; Roccheri, Maria Carmela

2016-01-01

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.

Review of NCRP radiation dose limit for embryo and fetus in occupationally-exposed women - approved 1977

International Nuclear Information System (INIS)

Anon.

1978-01-01

On the basis of the current review, the NCRP has decided to make no change in the current recommendation of its radiation dose limit to the unborn. The NCRP recommendation is restated here as follows: During the entire gestation period, the maximum permissible dose equivalent to the embryo-fetus from occupational exposure of the expectant mother should be 0.5 rem. Since the preparation of the 1971 report there has been no new evidence concerning teratogenic or carcinogenic effects of irradiation of the embryo-fetus that would justify a change in the limit in either direction. It is implicit in this position and recommendation that women who can reasonably be expected to be pregnant should not, in certain instances, be exposed to the same radiation environment as women who are not considered fertile or as men. This applies particularly to conditions where radiation workers can receive dose equivalents of 0.5 rem or more in short periods

Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

Science.gov (United States)

Rascalou, Adeline; Lamartine, Jérôme; Poydenot, Pauline; Demarne, Frédéric; Bechetoille, Nicolas

2018-05-05

Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm 2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede

Enhanced malignant transformation is accompanied by increased survival recovery after ionizing radiation in Chinese hamster embryo fibroblasts

International Nuclear Information System (INIS)

Boothman, D.A.

1994-01-01

Transformed Chinese hamster embryo fibroblasts (CHEF), which gradually increase in tumor-forming ability in nude mice, were isolated from normal diploid CHEF/18 cells. Transformed CHEF cells (i.e., T30-4 > 21-2M3 > 21-2 > normal CHEF/18) showed gradual increases in potentially lethal damage (PLD) survival recovery. β-Lapachone and camptothecin, modulators of topoisomerase I (Topo I) activity, not only prevented survival recovery in normal as well as in tumor cells, but enhanced unscheduled DNA synthesis. These seemingly conflicting results are due to the fact that Topo I activity can be modulated by inhibitors to convert single-stranded DNA lesions into double-stranded breaks. Increases in unscheduled DNA synthesis may result from a continual supply of free ends, on which DNA repair processes may act. Altering Topo I activity with modulators appears to increase X-ray lethality via a DNA lesion modification suicide pathway. Cells down-regulate Topo I immediately after ionizing radiation to prevent Topo I-mediated lesion modification and to enhance survival recovery. 16 refs., 3 figs., 1 tab

Transcriptional Profiling of Host Gene Expression in Chicken Embryo Fibroblasts Infected with Reticuloendotheliosis Virus Strain HA1101.

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Ji Miao

Full Text Available Reticuloendotheliosis virus (REV, a member of the Gammaretrovirus genus in the Retroviridae family, causes an immunosuppressive, oncogenic and runting-stunting syndrome in multiple avian hosts. To better understand the host interactions at the transcriptional level, microarray data analysis was performed in chicken embryo fibroblast cells at 1, 3, 5, and 7 days after infection with REV. This study identified 1,785 differentially expressed genes that were classified into several functional groups including signal transduction, immune response, biological adhesion and endocytosis. Significant differences were mainly observed in the expression of genes involved in the immune response, especially during the later post-infection time points. These results revealed that differentially expressed genes IL6, STAT1, MyD88, TLRs, NF-κB, IRF-7, and ISGs play important roles in the pathogenicity of REV infection. Our study is the first to use microarray analysis to investigate REV, and these findings provide insights into the underlying mechanisms of the host antiviral response and the molecular basis of viral pathogenesis.

Alteration of neurotransmission and skeletogenesis in sea urchin Arbacia lixula embryos exposed to copper oxide nanoparticles.

Science.gov (United States)

Cappello, Tiziana; Vitale, Valeria; Oliva, Sabrina; Villari, Valentina; Mauceri, Angela; Fasulo, Salvatore; Maisano, Maria

2017-09-01

The extensive use of copper oxide nanoparticles (CuO NPs) in many applications has raised concerns over their toxicity on environment and human health. Herein, the embryotoxicity of CuO NPs was assessed in the black sea urchin Arbacia lixula, an intertidal species commonly present in the Mediterranean. Fertilized eggs were exposed to 0.7, 10 and 20ppb of CuO NPs, until pluteus stage. Interferences with the normal neurotransmission pathways were observed in sea urchin embryos. In detail, evidence of cholinergic and serotoninergic systems affection was revealed by dose-dependent decreased levels of choline and N-acetyl serotonin, respectively, measured by nuclear magnetic resonance (NMR)-based metabolomics, applied for the first time to our knowledge on sea urchin embryos. The metabolic profile also highlighted a significant CuO NP dose-dependent increase of glycine, a component of matrix proteins involved in the biomineralization process, suggesting perturbed skeletogenesis accordingly to skeletal defects in spicule patterning observed previously in the same sea urchin embryos. However, the expression of skeletogenic genes, i.e. SM30 and msp130, did not differ among groups, and therefore altered primary mesenchyme cell (PMC) migration was hypothesized. Other unknown metabolites were detected from the NMR spectra, and their concentrations found to be reflective of the CuO NP exposure levels. Overall, these findings demonstrate the toxic potential of CuO NPs to interfere with neurotransmission and skeletogenesis of sea urchin embryos. The integrated use of embryotoxicity tests and metabolomics represents a highly sensitive and effective tool for assessing the impact of NPs on aquatic biota. Copyright © 2017 Elsevier Inc. All rights reserved.

DsRed gene expression by doxycycline in porcine fibroblasts and ...

African Journals Online (AJOL)

DsRed gene expression by doxycycline in porcine fibroblasts and cloned embryos using transposon. SuJin Kim, JoonHo Moon, BegoRoibas da Torre, Islam M Saadeldin, JungTaek Kang, JiYei Choi, SolJi Park, Byeong-Chun Lee, Goo Jang Goo Jang ...

Effect of quercetin on the number of blastomeres, zona pellucida thickness, and hatching rate of mouse embryos exposed to actinomycin D: An experimental study

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Hamid Reza Sameni

2018-02-01

Full Text Available 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.

Proteomic analysis of zebrafish embryos exposed to simulated-microgravity

Science.gov (United States)

Hang, Xiaoming; Ma, Wenwen; Wang, Wei; Liu, Cong; Sun, Yeqing

Microgravity can induce a serial of physiological and pathological changes in human body, such as cardiovascular functional disorder, bone loss, muscular atrophy and impaired immune system function, etc. In this research, we focus on the influence of microgravity to vertebrate embryo development. As a powerful model for studying vertebrate development, zebrafish embryos at 8 hpf (hour past fertilization) and 24 hpf were placed into a NASA developed bioreac-tor (RCCS) to simulate microgravity for 64 and 48 hours, respectively. The same number of control embryos from the same parents were placed in a tissue culture dish at the same temper-ature of 28° C. Each experiment was repeated 3 times and analyzed by two-dimensional (2-D) gel electrophoresis. Image analysis of silver stained 2-D gels revealed that 64 from total 292 protein spots showed quantitative and qualitative variations that were significantly (P<0.05) and reproducibly different between simulate-microgravity treatment and the stationary control samples. 4 protein spots with significant expression alteration (P<0.01) were excised from 2-D gels and analyzed by MALDI-TOF/TOF mass spectra primarily. Of these proteins, 3 down-regulated proteins were identified as bectin 2, centrosomal protein of 135kDa and tropomyosin 4, while the up-regulated protein was identified as creatine kinase muscle B. Other protein spots showed significant expression alteration will be identified successively and the corresponding genes expression will also be measured by Q-PCR method at different development stages. The data presented in this study illustrate that zebrafish embryo can be significantly induced by microgravity on the expression of proteins involved in bone and muscle formation. Key Words: Danio rerio; Simulated-microgravity; Proteomics

Membrane associated ion transport enzymes in normal and transformed fibroblasts and epithelial cells

International Nuclear Information System (INIS)

Borek, C.

1982-01-01

In an effort to evaluate membrane changes associated with neoplastic transformation of fibroblasts and epithelial cells by radiation and chemicals, alterations in membrane-associated (Na + + K + )-ATPase and 5'-nucleotidase activities were investigated. Cell cultures consisted of normal and radiation transformed hamster embryo fibroblasts (HE) and mouse C3H 10T 1/2 fibroblasts, normal and chemically transformed adult rat liver epithelial cells (ARL), as well as hepatocarcinoma cells induced by the liver transformants. Transformed fibroblasts demonstrated a 1-2 fold increase in (Na + + K + )-ATPase activity over the normal, while the transformed liver epithelial cells and carcinoma cells showed a 60% and 40% decrease in activity compared to the normal values, respectively. The 5'-nucleotidase activity was 2 to 3 times higher in the transformed fibroblasts

Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes

International Nuclear Information System (INIS)

Lee, Eugine; Lee, So Hyun; Kim, Sue

2006-01-01

Xenotransplantation is a rapidly expanding field of research and cloned miniature pigs have been considered as a model animal for it. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several kinds of aberrant development. A possible explanation for the developmental failure of SCNT embryos is insufficient reprogramming of the somatic cell nucleus by the oocyte. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and embryonic germ cell nuclei with Oct-4 and Oct-4 related genes (Ndp5211, Dppa2, Dppa3, and Dppa5), which are important for embryonic development, Hand1 and GATA-4, which are important for placental development, as molecular markers using RT-PCR. The Oct-4 expression level was significantly lower (P < 0.05) in cloned hatched blastocysts derived from fibroblasts and many of fibroblast-derived clones failed to reactivate at least one of the tested genes, while most of the germ cell clones and control embryos correctly expressed these genes. In conclusion, our results suggest that the reprogramming of fibroblast-derived cloned embryos is highly aberrant and this improper reprogramming could be one reason of the early lethality and post-implantation anomalies of somatic cell-derived clones

Toxicity of Prudhoe Bay crude oil to mallard duck (Anas Platyrhynchos) embryos

International Nuclear Information System (INIS)

Lusimbo, W.S.

1999-01-01

This thesis examined the rates and timing of mortality and hatchability of mallard duck embryos that have been exposed to Prudhoe Bay crude oil (PBCO). The objective was to identify any pathological abnormalities that may suggest that the chorioallantoic membrane (CAM) is a target organ of toxicity. The study involved the external application of PBCO to the eggshell to mimic the natural route of exposure. Embryo mortality was then determined at different days of incubation and the most sensitive age of incubation to oil was established. On the basis of initial results, the following 3 hypothesis were formulated: (1) the CAM is a target organ of oil toxicity, (2) injury to the CAM compromises its physiological role of calcium mobilization from the eggshell, and (3) oil exposure causes anemia and damage to red blood cells of mallard embryos. Data was compared with data reported in chicken embryos exposed to the same type of petroleum oil. It was shown that the toxic effects of PBCO to mallard duck embryos were similar to those found in chicken embryos. Oil-exposed embryos exhibited high mortality and reduced growth. Pathological changes in liver, kidneys and bursa of Fabricius, in oil-exposed mallard embryos were also similar to those of exposed chicken embryos. It was noted that this is the first study to recognize and describe pathological changes in the chorioallantoic membrane (CAM) of avian embryos exposed to petroleum oil. Lesions in the CAM were found in areas right beneath the oil on the egg shell and in areas distant from any such direct contact, suggesting that direct contact with the oil was not the main cause of injury to the CAM. The occurrence of lesions in CAM were highest immediately following exposure to PBCO. The lesions observed suggest two different kids of toxic effects, lethal injury to cells in various tissues, and alteration in the timing of organ development. The author notes that more remains to be learned regarding the toxic effects of

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

DEFF Research Database (Denmark)

Svarcova, Olga; Dinnyes, A.; Polgar, Z.

2009-01-01

displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

Effect of Fibroblast Growth Factor 2 (FGF2 and Insulin Transferrin Selenium (ITS on In Vitro Maturation, Fertilization and Embryo Development in Sheep

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Sukanta Mondal

2015-08-01

Full Text Available The present study evaluated the effect of fibroblast growth factor 2 (FGF2 and insulin-transferrin-selenium (ITS to the in vitro maturation and embryo culture media on ovine oocyte maturation, cleavage and embryo development. Oocytes having more than five layers of unexpanded cumulus cells and granular homogenous ooplasm were cultured into 50 μL droplets of eight different culture systems: (i TCM-199 (Tissue Culture Medium-199; (ii TCM-199+10 ng/mL FGF2; (iii TCM-199+20 ng/mL FGF2; (iv TCM-199+30 ng/mL FGF2; (v TCM-199+10 ng/mL ITS; (vi TCM-199+20 ng/mL ITS; (vii TCM-199+30 ng/mL ITS and (viii TCM-199+20 ng/mL ITS+20 ng/mL FGF2 in a CO2 incubator at 38.50C for 24 h. All the oocyte culture media were supplemented with 10% FBS, FSH (10 μg/mL and gentamicin (50 µg/mL. The maturation rate was assessed based on the degree of expansion of cumulus cells and identifying first polar body extrusion into perivitelline space. The matured oocytes were inseminated with 1 to 2 million spermatozoa/mL in Brackett and Oliphant medium and the cleavage rate was checked after 42-48 h post insemination and further cultured for 6-7 days. Maturation and cleavage rates were significantly higher (P<0.05 in the oocytes cultured in TCM-199 +10% FBS+FSH (10 μg/mL supplemented with both 20 ng/mL ITS and 20 ng/mL FGF2 as compared to the control. It was concluded that the supplementation of ITS and FGF2 in maturation medium was beneficial for improving maturation and cleavage rates of sheep oocytes. The addition of ITS and FGF2 in embryo culture medium did not improve the development of sheep embryos.

Viability of quail embryos and nestlings from the eggs exposed to gamma-radiation, vibration and storage

International Nuclear Information System (INIS)

Mishchenko, V.F.; Shafirkin, A.V.

1986-01-01

The viability of quail embryos and nestlings from the incubation eggs exposed (in) to gamma-radiation at a dose of 300 cGy and stored for 15 days or (ii) to gamma-radiation at a dose of 300 cGy and stored for 30 days or (iii) to vibration with the acute egg end oriented contrary to the vibration front and stored for 30 days did not decrease as compared to that of the controls stored for the same time period. The viability diminished if the radiation dose was increased to 600-1200 cGy of if the egg orientation during vibration was changed

Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

DEFF Research Database (Denmark)

Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus

2002-01-01

A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity...

Induction of chromosomal aberrations in human primary fibroblasts and immortalized cancer cells exposed to extremely-low-frequency electromagnetic fields

International Nuclear Information System (INIS)

Seyyedi, S. S.; Mozdarani, H.; Rezaei Tavirani, M.; Heydari, S.

2010-01-01

Rapidly increasing possibilities of exposure to environmental extremely low-frequency electromagnetic fields have become a topic of worldwide investigation. Epidemiological and laboratory studies suggest that exposure to extremely low-frequency electromagnetic fields may increase cancer risk therefore assessment of chromosomal damage in various cell lines might be of predictive value for future risk estimation. Materials and Methods: Primary cultures of fibroblasts from human skin biopsy were exposed to continuous extremely low-frequency electromagnetic fields (3, 50 and 60 Hz, sinusoidal, 3h, and 4 m T). Also immortalized cell lines, SW480, MCF-7 and 1321N1 were exposed to continuous extremely low-frequency electromagnetic fields (50 Hz, sinusoidal, 3 h, 4 m T). Metaphase plates Were prepared according to standard methods and stained in 5% Giemsa solution. Chromosomal aberrations of both chromosome and chromatid types were scored to evaluate the effects of extremely low-frequency electromagnetic fields on primary or established cell lines. Results: Results indicate that by increasing the frequency of extremely low-frequency electromagnetic fields, chromosomal aberrations were increased up to 7-fold above background levels in primary human fibroblast cells. In addition, continuous exposure to a 50 Hz electromagnetic field led to a significant increase in chromosomal aberrations in SW480, MCF-7 and 1321N1 cell lines compared to sham control. Conclusion: Results obtained indicate that extremely low-frequency electromagnetic fields has the potential for induction of chromosomal aberrations in all cell types.

Assessment of DNA Damage by RAPD in Paracentrotus lividus Embryos Exposed to Amniotic Fluid from Residents Living Close to Waste Landfill Sites

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Maurizio Guida

2010-01-01

Full Text Available The aim of this study was to assess the genotoxic effects of environmental chemicals on residents living near landfills. The study was based on samples of amniotic fluid from women living in the intensely polluted areas around the Campania region of Italy compared to a nonexposed control group. We evaluated the genetic effects that this amniotic fluids collected in contaminated sites had on Paracentrotus lividus embryos. DNA damage was detected through changes in RAPD (Random Amplified Polymorphism DNA profiles. The absence of the amplified DNA fragments indicated deletions in Paracentrotus lividus DNA exposed to the contaminated amniotic fluids when compared to equal exposure to uncontaminated fluids. These results show the ability of RAPD-PCR to detect and isolate DNA sequences representing genetic alterations induced in P. lividus embryos. Using this method, we identified two candidate target regions for DNA alterations in the genome of P. lividus. Our research indicates that RAPD-PCR in P. lividus embryo DNA can provide a molecular approach for studying DNA damage from pollutants that can impact human health. To our knowledge, this is the first time that assessment of DNA damage in P. lividus embryos has been tested using the RAPD strategy after exposure to amniotic fluid from residents near waste landfill sites.

Differential expression of Toll-like receptor pathway genes in chicken embryo fibroblasts from chickens resistant and susceptible to Marek's disease.

Science.gov (United States)

Haunshi, Santosh; Cheng, Hans H

2014-03-01

The Toll-like receptor (TLR) signaling pathway is one of the innate immune defense mechanisms against pathogens in vertebrates and invertebrates. However, the role of TLR in non-MHC genetic resistance or susceptibility to Marek's disease (MD) in the chicken is yet to be elucidated. Chicken embryo fibroblast (CEF) cells from MD susceptible and resistant lines were infected either with Marek's disease virus (MDV) or treated with polyionosinic-polycytidylic acid, a synthetic analog of dsRNA, and the expression of TLR and pro-inflammatory cytokines was studied at 8 and 36 h posttreatment by quantitative reverse transcriptase PCR. Findings of the present study reveal that MDV infection and polyionosinic-polycytidylic acid treatment significantly elevated the mRNA expression of TLR3, IL6, and IL8 in both susceptible and resistant lines. Furthermore, basal expression levels in uninfected CEF for TLR3, TLR7, and IL8 genes were significantly higher in resistant chickens compared with those of susceptible chickens. Our results suggest that TLR3 together with pro-inflammatory cytokines may play a significant role in genetic resistance to MD.

Evaluation of median survival patients with acute lymphoblastic leukemia exposed ionizing radiation by the Chornobyl accident, depending on the aminoacid composition of stromal fibroblasts bone marrow and indicators of myelogram

International Nuclear Information System (INIS)

Bebeshko, V.G.; Bruslova, K.M.; Tsvjetkova, N.M.; Pushkar'ova, T.Yi.; Panchenko, L.M.; Yatsemirs'kij, S.N.; Volodyina, T.T.; Lyashenko, L.O.

2015-01-01

The aminoacid composition in the stromal fibroblasts of bone marrow and indexes of hemogram in children with acute lymphoblastic leukemias, which exposed to radiation from the Chornobyl accident, for evaluation of the median survival was investigated. In the study 54 patients with acute lymphoblastic leukemia, residents of Kyiv, Zhytomyr and Chernihiv regions of Ukraine were involved. Effectiveness of stromal fibroblasts colony forming units of bone marrow and aminoacid composition of fibroblasts were studied. Bone marrow examination was conducted on day 33 and after stop of chemotherapy, the median survival of patients and exposure dose were evaluated. In children with acute leukemias effectiveness of stromal fibroblasts of bone marrow was 2-2.3 times lower than normative. Correlation (r_s = +0.83) between the number of erythroid progenitor cells in the bone marrow and proline content in stromal fibroblasts established . The correlation between patients median survival and number of granulocytes in myelogram (r_s = + 0.82) was proven. Mean exposure doses in patients was (5.86 ±1.11) mSv and it did not correlate with myelogram results, effectiveness of stromal fibroblasts of bone marrow and median survival. The median survival of patients with acute leukemias depends on the content of amino acids in stromal fibroblasts of bone marrow and recover of hematopoiesis after chemotherapy

Birth of cloned calves from vitrified-warmed zona-free buffalo (Bubalus bubalis) embryos produced by hand-made cloning.

Science.gov (United States)

Saha, Ambikaprasanna; Panda, Sudeepta K; Chauhan, Manmohan S; Manik, Radhey S; Palta, Prabhat; Singla, Suresh K

2013-01-01

The availability of techniques for the vitrification of cloned blastocysts can improve their effective use. The present study compared the developmental competence of buffalo cloned embryos derived from adult (BAF), newborn (BNF) and fetal fibroblast (BFF) before and after vitrification. Despite similar cleavage rates among the three groups, the blastocyst rate was lower for BAF- than BNF- and BFF-derived embryos (30.2±2.2% vs 41.7±1.7% and 39.1±2.1%, respectively; Pcloned buffalo embryos cryopreserved by vitrification can be used to obtain live offspring.

Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos.

Science.gov (United States)

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Yao, Chaogang; Zhou, Yang; Zhu, Jianguo; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

2011-12-01

Valproic acid (VPA), a histone deacetylase inbibitor, has been shown to generate inducible pluripotent stem (iPS) cells from mouse and human fibroblasts with a significant higher efficiency. Because successful cloning by somatic cell nuclear transfer (SCNT) undergoes a full reprogramming process in which the epigenetic state of a differentiated donor nuclear is converted into an embryonic totipotent state, we speculated that VPA would be useful in promoting cloning efficiency. Therefore, in the present study, we examined whether VPA can promote the developmental competence of SCNT embryos by improving the reprogramming state of donor nucleus. Here we report that 1 mM VPA for 14 to 16 h following activation significantly increased the rate of blastocyst formation of porcine SCNT embryos constructed from Landrace fetal fibroblast cells compared to the control (31.8 vs. 11.4%). However, we found that the acetylation level of Histone H3 lysine 14 and Histone H4 lysine 5 and expression level of Oct4, Sox2, and Klf4 was not significantly changed between VPA-treated and -untreated groups at the blastocyst stage. The SCNT embryos were transferred to 38 surrogates, and the cloning efficiency in the treated group was significantly improved compared with the control group. Taken together, we have demonstrated that VPA can improve both in vitro and in vivo development competence of porcine SCNT embryos.

Acute toxicity and gene responses induced by endosulfan in zebrafish (Danio rerio embryos

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Young-Sun Moon

2016-10-01

Full Text Available Endosulfan has been listed as a persistent organic pollutant, and is frequently found in agricultural environments during monitoring processes owing to its heavy use and persistent characteristics. This study was conducted to understand the effects of endosulfan on the development of zebrafish (Danio rerio embryos by exposing them to a specific range of endosulfan concentrations. Exposing zebrafish embryos to endosulfan for 96 h yielded no acute toxicity until the concentration reached 1500 μg L−1, whereas malformed zebrafish larvae developed severely curved spines and shortened tails. About 50% of zebrafish larvae were malformed when exposed to 600 μg L−1 of endosulfan. Comparative gene expression using real-time quantitative polymerase chain reaction was assessed using endosulfan-exposed zebrafish embryos. CYP1A and CYP3A were significantly enhanced in response to endosulfan treatment. Two genes, acacb and fasn, encoding acetyl-CoA carboxylase b and fatty acid synthase proteins, respectively, were also up-regulated after treating zebrafish embryos with endosulfan. These genes are also involved in fatty acid biosynthesis. The genes encoding vitellogenin and Hsp70 increased in a concentration-dependent manner in embryos. Finally, biochemical studies showed that acetylcholinesterase activity was reduced, whereas glutathione S-transferase and carboxylesterase activities were enhanced in zebrafish embryos after endosulfan treatment. These biochemical and molecular biological differences might be used for tools to determine contamination of endosulfan in the aquatic environment.

Production of a cloned calf from a fetal fibroblast cell line

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Mello M.R.B.

2003-01-01

Full Text Available The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5% were successfully fused and 24 (28.9% developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8% of which were pregnant on day 35 and 3 (16.7% on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg and sexual behavior (libido and semen characteristics.

The constitutive activation of the CEF-4/9E3 chemokine gene depends on C/EBPbeta in v-src transformed chicken embryo fibroblasts

DEFF Research Database (Denmark)

Gagliardi, M; Maynard, S; Bojovic, B

2001-01-01

The CEF-4/9E3 chemokine gene is expressed constitutively in chicken embryo fibroblasts (CEF) transformed by the Rous sarcoma virus (RSV). This aberrant induction is controlled at the transcriptional and post-transcriptional levels. Transcriptional activation depends on multiple elements of the CEF....../EBPbeta binds to a second element located in proximity of the TRE. A mutation of this distal CAAT box impaired the activation of the CEF-4 promoter by pp60(v-src) indicating that this element is also part of the SRU. Using the RCASBP retroviral vector, we expressed a dominant negative mutant of C....../EBPbeta (designated Delta184-C/EBPbeta) in RSV-transformed CEF. Delta184-C/EBPbeta decreased the accumulation of the CEF-4 mRNA and activation of the CEF-4 promoter by pp60(v-src). The induction of the Cox-2 gene (CEF-147) was also reduced by Delta184-C/EBPbeta. The effect of the dominant negative mutant was observed...

Connective tissue fibroblasts and Tcf4 regulate myogenesis

Science.gov (United States)

Mathew, Sam J.; Hansen, Jody M.; Merrell, Allyson J.; Murphy, Malea M.; Lawson, Jennifer A.; Hutcheson, David A.; Hansen, Mark S.; Angus-Hill, Melinda; Kardon, Gabrielle

2011-01-01

Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis. PMID:21177349

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

Science.gov (United States)

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them.

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Sushil Kumar Mohapatra

Full Text Available Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT has had a limited applicability due to very low (>1% live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF and Hand-made cloning (TE-HMC, and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF.

Establishment of Trophectoderm Cell Lines from Buffalo (Bubalus bubalis) Embryos of Different Sources and Examination of In Vitro Developmental Competence, Quality, Epigenetic Status and Gene Expression in Cloned Embryos Derived from Them

Science.gov (United States)

Mohapatra, Sushil Kumar; Sandhu, Anjit; Singh, Karn Pratap; Singla, Suresh Kumar; Chauhan, Manmohan Singh; Manik, Radheysham; Palta, Prabhat

2015-01-01

Despite being successfully used to produce live offspring in many species, somatic cell nuclear transfer (NT) has had a limited applicability due to very low (>1%) live birth rate because of a high incidence of pregnancy failure, which is mainly due to placental dysfunction. Since this may be due to abnormalities in the trophectoderm (TE) cell lineage, TE cells can be a model to understand the placental growth disorders seen after NT. We isolated and characterized buffalo TE cells from blastocysts produced by in vitro fertilization (TE-IVF) and Hand-made cloning (TE-HMC), and compared their growth characteristics and gene expression, and developed a feeder-free culture system for their long-term culture. The TE-IVF cells were then used as donor cells to produce HMC embryos following which their developmental competence, quality, epigenetic status and gene expression were compared with those of HMC embryos produced using fetal or adult fibroblasts as donor cells. We found that although TE-HMC and TE-IVF cells have a similar capability to grow in culture, significant differences exist in gene expression levels between them and between IVF and HMC embryos from which they are derived, which may have a role in the placental abnormalities associated with NT pregnancies. Although TE cells can be used as donor cells for producing HMC blastocysts, their developmental competence and quality is lower than that of blastocysts produced from fetal or adult fibroblasts. The epigenetic status and expression level of many important genes is different in HMC blastocysts produced using TE cells or fetal or adult fibroblasts or those produced by IVF. PMID:26053554

Induction of SCE by DNA cross-links in human fibroblasts exposed to 8-MOP and UVA irradiation

International Nuclear Information System (INIS)

Bredberg, A.; Lambert, B.

1983-01-01

To study the SCE-inducing effect of psoralen cross-links in the DNA of normal, human fibroblasts, cell cultures were exposed to PUVA (0.2-1 μg of 8-MOP per ml, followed by UVA irradiation at 0.04 J/cm 2 ) and carefully washed to remove non-covalently bound psoralen. Some cell cultures were then given a second dose of UVA (1.1 J/cm 2 ), either immediately after PUVA or 1-3 days later. By this type of treatment, cells with different proportions of DNA cross-links are obtained. The initial PUVA treatment will mainly give rise to psoralen monoadducts and only few cross-links in the DNA, and the second UVA irradiation will convert a number of the psoralen monoadducts into cross-links. (orig./AJ)

Dealing of embryo/fetal exposures in a medical setting

International Nuclear Information System (INIS)

Miller, K.L.

1991-01-01

The medical environment presents two uniquely different situations involving potential radiation exposure to the embryo or fetus. On the one hand we have the potential for radiation exposure to the embryo of fetus of the occupationally exposed employee where the recommendations are well defined. On the other hand we have the pregnant or potentially pregnant patient requiring diagnostic radiological examinations or therapeutic procedures where the recommendations are not as well defined. This paper provides an overview of both situations, a discussion of potential exposures and a review of methods for estimating fetal doses when the exposed individual is the patient

Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

Science.gov (United States)

Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

2015-01-01

Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors.

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Ramiro Olivera

Full Text Available The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647 on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I induced pluripotent stem cells (iPSCs vs. adult fibroblasts (AF fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II umbilical cord-derived mesenchymal stromal cells (UC-MSCs vs. fetal fibroblasts derived from an unborn cloned foetus (FF vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively, respect to <1h (5.2% and 2%, respectively and 1-2h (5.6% and 4.7%, respectively groups (P<0.05. However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2% or 1-2h (35.7% were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6% than using FF (8.9% or AF (9.3%, (P<0.05. Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively, viable foals (two were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals.

Six cloned calves produced from adult fibroblast cells after long-term culture

Science.gov (United States)

Kubota, Chikara; Yamakuchi, Hiroshi; Todoroki, Junichi; Mizoshita, Kazunori; Tabara, Norio; Barber, Michele; Yang, Xiangzhong

2000-01-01

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as “gene knock-out” by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10–15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10–12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells. PMID:10655472

Toxicity of beauvericin on porcine oocyte maturation and preimplantation embryo development

NARCIS (Netherlands)

Schoevers, Eric J; Santos, Regiane R; Fink-Gremmels, Johanna; Roelen, Bernard A J

2016-01-01

Beauvericin (BEA) is one of many toxins produced by Fusarium species that contaminate feed materials. The aim of this study was to assess its effects on porcine oocyte maturation and preimplantation embryo development. Cumulus-oocyte-complexes and developing embryos were exposed to BEA and cultured

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos

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Estelle H. Venter

2011-02-01

Full Text Available Bluetongue (BT, a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV, can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 103.5 respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

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Humidah Alanazi

2014-01-01

Full Text Available The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P<0.01 sensitive to oxidation but significantly (P<0.01 resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P<0.01 slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

In vivo and in vitro development of Tibetan antelope (Pantholops hodgsonii interspecific cloned embryos

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Guanghua SU,Lei CHENG,Yu GAO,Kun LIU,Zhuying WEI,Chunling BAI,Fengxia YIN,Li GAO,Guangpeng LI,Shorgan BOU

2014-02-01

Full Text Available The Tibetan antelope is endemic to the Tibetan Plateau, China, and is now considered an endangered species. As a possible rescue strategy, the development of embryos constructed by interspecies somatic cell nuclear transfer (iSCNT was examined. Tibetan antelope fibroblast cells were transferred into enucleated bovine, ovine and caprine oocytes. These cloned embryos were then cultured in vitro or in the oviducts of intermediate animals. Less than 0.5% of the reconstructed antelope-bovine embryos cultured in vitro developed to the blastocyst stage. However, when the cloned antelope-bovine embryos were transferred to caprine oviducts, about 1.6% of the embryos developed to the blastocyst stage. In contrast, only 0.7% of the antelope-ovine embryos developed to the morula stage and none developed to blastocysts in ovine oviducts. The treatment of donor cells and bovine oocytes with trichostatin A did not improve the embryo development even when cultured in the oviducts of ovine and caprine. When the antelope-bovine embryos, constructed from oocytes treated with roscovitine or trichostatin A, were cultured in rabbit oviducts 2.3% and 14.3% developed to blastocysts, respectively. It is concluded that although some success was achieved with the protocols used, interspecies cloning of Tibetan antelope presents difficulties still to be overcome. The mechanisms resulting in the low embryo development need investigation and progress might require a deeper understanding of cellular reprogramming.

Adaptation to osmotic stress provides protection against ammonium nitrate in Pelophylax perezi embryos

International Nuclear Information System (INIS)

Ortiz-Santaliestra, Manuel E.; Fernandez-Beneitez, Maria Jose; Lizana, Miguel; Marco, Adolfo

2010-01-01

The negative effects of pollution on amphibians are especially high when animals are additionally stressed by other environmental factors such as water salinity. However, the stress provoked by salinity may vary among populations because of adaptation processes. We tested the combined effect of a common fertilizer, ammonium nitrate (0-90.3 mg N-NO 3 NH 4 /L), and water salinity (0-2 per mille ) on embryos of two Pelophylax perezi populations from ponds with different salinity concentrations. Embryos exposed to the fertilizer were up to 17% smaller than controls. Survival rates of embryos exposed to a single stressor were always below 10%. The exposure to both stressors concurrently increased mortality rate (>95%) of embryos from freshwater. Since the fertilizer was lethal only when individuals were stressed by the salinity, it did not cause lethal effects on embryos naturally adapted to saline environments. Our results underscore the importance of testing multiple stressors when analyzing amphibian sensitivity to environmental pollution. - Natural resistance to salinity minimizes the impact of chemical fertilizers on amphibian embryos.

Predator recognition in rainbowfish, Melanotaenia duboulayi, embryos.

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Lois Jane Oulton

Full Text Available Exposure to olfactory cues during embryonic development can have long term impacts on birds and amphibians behaviour. Despite the vast literature on predator recognition and responses in fishes, few researchers have determined how fish embryos respond to predator cues. Here we exposed four-day-old rainbowfish (Melanotaenia duboulayi embryos to cues emanating from a novel predator, a native predator and injured conspecifics. Their response was assessed by monitoring heart rate and hatch time. Results showed that embryos have an innate capacity to differentiate between cues as illustrated by faster heart rates relative to controls. The greatest increase in heart rate occurred in response to native predator odour. While we found no significant change in the time taken for eggs to hatch, all treatments experienced slight delays as expected if embryos are attempting to reduce exposure to larval predators.

Mechanisms of cadmium-caused eye hypoplasia and hypopigmentation in zebrafish embryos

International Nuclear Information System (INIS)

Zhang, Ting; Zhou, Xin-Ying; Ma, Xu-Fa; Liu, Jing-Xia

2015-01-01

Highlights: Using high-throughput in situ hybridization screening, we found that genes labeling the neural crest and its derivative pigment cells were sensitive to cadmium toxicity during zebrafish organogenesis, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in cadmium-exposed embryos. Based on neural crest markers, we identified the doses and times of cadmium exposure that cause damage to the zebrafish organogenesis, and we also found that compounds BIO or RA could neutralize the toxic effects of cadmium. - Abstract: Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24 hpf, when cadmium (Cd) dosage was above 17.8 μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24 hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd.

Mechanisms of cadmium-caused eye hypoplasia and hypopigmentation in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Zhang, Ting, E-mail: zting@webmail.hzau.edu.cn; Zhou, Xin-Ying, E-mail: 290356082@qq.com; Ma, Xu-Fa, E-mail: xufama@mail.hzau.edu.cn; Liu, Jing-Xia, E-mail: ichliu@mail.hzau.edu.cn

2015-10-15

Highlights: Using high-throughput in situ hybridization screening, we found that genes labeling the neural crest and its derivative pigment cells were sensitive to cadmium toxicity during zebrafish organogenesis, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in cadmium-exposed embryos. Based on neural crest markers, we identified the doses and times of cadmium exposure that cause damage to the zebrafish organogenesis, and we also found that compounds BIO or RA could neutralize the toxic effects of cadmium. - Abstract: Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24 hpf, when cadmium (Cd) dosage was above 17.8 μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24 hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd.

Mental retardation occurring in embryo exposed in utero to the atomic bomb (Hiroshima). After 30 years follow-up study

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Ishikawa, H; Shimasaki, A; Fujiwara, K [Saijo Mental Hospital, Hiroshima (Japan); Harada, M; Minami, R

1978-11-01

This paper deals with a long term follow-up study on psychological symptoms in four patients with microscopically microcephaly induced by prenatal exposed to atomic bomb. They were exposed to atomic bomb at 8- and 12-week-embryos. The distance from the center of the explosion was 780 - 1180 m. All their mothers had acute radiation hazards. Their growths in the uterus were markedly damaged. Postnatal body weight ranged between 1,300 and 2,000 g. They were commonly characterized by microcephaly, physiological and mental retardation, stigmata of degeneracy, and skin symptoms, who were diagnosed as ''microcephaly induced by early prenatal exposure to atomic bomb'' by the research group for microcephaly in the Ministry of Health and Welfare. These common symptoms such as microcephaly, stigmata of degeneracy, and disturbed growth, but neurological symptoms such as motor aphasia were slighter in these patients than in those having congenital Minamata disease prenatal. These results suggested that the prognosis of these patients in whom 30 years have passed is unexpectedly serious.

Survival, Fertilization and Developmental Rates of Cryotop-Vitrified Oocyte and Embryo Using Low Concentrated Cryoprotectants

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A Roozbehi

2012-10-01

Full Text Available Background & Aim: The preserving embryos, the risk of multiple pregnancies, the existence of factors in stimulated uterine cycle, are important forces in perfecting embryo cryopreservation. The aim of current study was to assess Survival, Fertilization and Developmental Rates (SRs, FRs, DRs of the mouse oocytes and embryos using cryotop and low concentrated cryoprotectants solutions. Methods: Mouse C57BL/6 oocytes and embryos were collected. Oocytes SRs, FRs, DRs were recorded after cryotop-vitrification/ warming. As well as comparing fresh oocytes and embryos, the data obtained from experimental groups (exp. applying 1.25, 1.0, and 0.75 Molar (M CPAs were analyzed in comparison to those of exp. adopting 1.5 M CPAs (largely-used concentration of EthylenGlycol (EG and Dimethylsulphoxide (DMSO. Results: The data of oocytes exposed to 1.25 M CPAs were in consistency with those exposed to 1.5 M and control group in terms of SR, FR and DR. As fewer concentrations were applied, the more decreased SRs, FRs and DRs were obtained from other experimental groups. The results of embryos were exposed to 1.25 M and 1.0 M was close to those vitrified with 1.5 M and fresh embryos. The results of 0.75 M concentrated CPAs solutions were significantly lower than those of control, 1.5 M and 1.0 M treated groups. Conclusion: CPAs limited reduction to 1.25 M and 1.0 M instead of using 1.5 M, for oocyte and embryo cryotop-vitrification procedure may be a slight adjustment.

Altered methanol embryopathies in embryo culture with mutant catalase-deficient mice and transgenic mice expressing human catalase

International Nuclear Information System (INIS)

Miller, Lutfiya; Wells, Peter G.

2011-01-01

The mechanisms underlying the teratogenicity of methanol (MeOH) in rodents, unlike its acute toxicity in humans, are unclear, but may involve reactive oxygen species (ROS). Embryonic catalase, although expressed at about 5% of maternal activity, may protect the embryo by detoxifying ROS. This hypothesis was investigated in whole embryo culture to remove confounding maternal factors, including metabolism of MeOH by maternal catalase. C57BL/6 (C57) mouse embryos expressing human catalase (hCat) or their wild-type (C57 WT) controls, and C3Ga.Cg-Catb/J acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 4 mg/ml MeOH or vehicle, and evaluated for functional and morphological changes. hCat and C57 WT vehicle-exposed embryos developed normally. MeOH was embryopathic in C57 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed and turning, whereas hCat embryos were protected. Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to C3H WT controls, suggesting that endogenous ROS are embryopathic. MeOH was more embryopathic in aCat embryos than WT controls, with reduced anterior neuropore closure and head length only in catalase-deficient embryos. These data suggest that ROS may be involved in the embryopathic mechanism of methanol, and that embryonic catalase activity may be a determinant of teratological risk.

Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

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Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

2012-01-01

Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

Developmental Toxicity of Dextromethorphan in Zebrafish Embryos/Larvae

Science.gov (United States)

Xu, Zheng; Williams, Frederick E.; Liu, Ming-Cheh

2012-01-01

Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of dextromethorphan during the embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the Phase I and Phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction (RT-PCR) analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. PMID:20737414

Mechanisms of cadmium-caused eye hypoplasia and hypopigmentation in zebrafish embryos.

Science.gov (United States)

Zhang, Ting; Zhou, Xin-Ying; Ma, Xu-Fa; Liu, Jing-Xia

2015-10-01

Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24 hpf, when cadmium (Cd) dosage was above 17.8 μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24 hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd. Copyright © 2015 Elsevier B.V. All rights reserved.

Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro.

Science.gov (United States)

Park, Hyun-Jin; Salem, Mabrouka; Semlali, Abdelhabib; Leung, Kai P; Rouabhia, Mahmoud

2017-07-01

We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

Science.gov (United States)

Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

2017-10-01

Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

Identification of the heart as the critical site of adenosine mediated embryo protection

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Greene Robert W

2010-05-01

Full Text Available Abstract Background Our understanding of the mechanisms that protect the developing embryo from intrauterine stress is limited. Recently, adenosine has been demonstrated to play a critical role in protecting the embryo against hypoxia via adenosine A1 receptors (A1ARs, which are expressed in the heart, nervous system, and other sites during development. However, the sites of A1AR action that mediate embryo protection are not known. To determine if the heart is a key site of adenosine-mediated embryo protection, A1ARs were selectively deleted in the embryonic heart using a Cre-LoxP system in which the alpha-myosin heavy chain promoter drives Cre-recombinase expression and excision of the A1AR gene from cardiomyocytes. Results With increasing exposure of maternal hypoxia (10% O2 from 48-96 hours beginning at embryonic day (E 8.5, embryo viability decreased in the cardiac-A1AR deleted embryos. 48 hours of hypoxia reduced embryonic viability by 49% in embryos exposed from E10.5-12.5 but no effect on viability was observed in younger embryos exposed to hypoxia from E8.5-10.5. After 72 hours of hypoxia, 57.8% of the cardiac-A1AR deleted embryos were either dead or re-absorbed compared to 13.7% of control littermates and after 96 hours 81.6% of cardiac-A1AR deleted embryos were dead or re-absorbed. After 72 hours of hypoxia, cardiac size was reduced significantly more in the cardiac-A1AR deleted hearts compared to controls. Gene expression analysis revealed clusters of genes that are regulated by both hypoxia and A1AR expression. Conclusions These data identify the embryonic heart as the critical site where adenosine acts to protect the embryo against hypoxia. As such these studies identify a previously unrecognized mechanism of embryo protection.

Toxicity of multi-walled carbon nanotubes, graphene oxide, and reduced graphene oxide to zebrafish embryos.

Science.gov (United States)

Liu, Xiao Tong; Mu, Xi Yan; Wu, Xiao Li; Meng, Li Xuan; Guan, Wen Bi; Ma, Yong Qiang; Sun, Hua; Wang, Cheng Ju; Li, Xue Feng

2014-09-01

This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO), and reduced graphene oxide (RGO), on zebrafish embryos. The 2-h post-fertilization (hpf) zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations (1, 5, 10, 50, 100 mg/L) for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations ls were evaluated. Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Antioxidant Activity of Ixora parviflora in a Cell/Cell-Free System and in UV-Exposed Human Fibroblasts

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Hsiu-Mei Chiang

2011-07-01

Full Text Available Polyphenols and flavonoids possess a variety of biological activities including antioxidant and anti-tumor activities. Ixora parviflora is a member of the flavonoid-rich Rubiaceae family of flowering plants and used as folk medicine in India. The aim of this study was to investigate the antioxidant activity of Ixora parviflora extract (IPE in a cell-free system and erythrocytes, and the ability of IPE to inhibit reactive oxygen species (ROS generation in human fibroblasts (Hs68 after ultraviolet (UV exposure. Various in vitro antioxidant assays were employed in this study. The extraction yield of IPE was 17.4 ± 3.9%, the total phenolic content of IPE was 26.2 μg gallic acid equivalent (GAE/mg leaves dry weight and the total flavonoids content was 54.2 ± 4.4 μg quercetin equvalent (QE/mg extract. The content of chlorogenic acid was 9.7 ± 1.2 mg/g extract. IPE at 1000 μg/mL exhibited a reducing capacity of 90.5 ± 0.6%, a 1,1-diphenyl-2-picrylhydrazy (DPPH radical scavenging activity of 96.0 ± 0.4%, a ferrous chelating activity of 72.2 ± 3.5%, a hydroxyl radical scavenging activity of 96.8 ± 1.4%, and a hydrogen peroxide scavenging activity of 99.5 ± 3.3%. IPE at 500 μg/mL also possessed inhibitory activity against 2,2'-azobis (2-methylpropionamidine dihydrochloride (AAPH-induced hemolysis of erythrocytes (89.4 ± 1.8% and resulted in a 52.9% reduction in ROS generation in UV-exposed fibroblasts. According to our findings, IPE is a potent antioxidant and a potential anti-photoaging agent.

Multistep change in epidermal growth factor receptors during spontaneous neoplastic progression in Chinese hamster embryo fibroblasts

International Nuclear Information System (INIS)

Wakshull, E.; Kraemer, P.M.; Wharton, W.

1985-01-01

Whole Chinese hamster embryo lineages have been shown to undergo multistep spontaneous neoplastic progression during serial passage in culture. The authors have studied the binding, internalization, and degradation of 125 I-labeled epidermal growth factor at four different stages of transformation. The whole Chinese hamster embryo cells lost cell surface epidermal growth factor receptors gradually during the course of neoplastic progression until only 10% of the receptor number present in the early-passage cells (precrisis) were retained in the late-passage cells (tumorigenic). No differences in internalization rates, chloroquine sensitivity, or ability to degrade hormone between the various passage levels were seen. No evidence for the presence in conditioned medium of transforming growth factors which might mask or down-regulate epidermal growth factor receptor was obtained. These results suggest that a reduction in cell surface epidermal growth factor receptor might be an early event during spontaneous transformation in whole Chinese hamster embryo cells

Propylthiouracil is teratogenic in murine embryos.

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Valeria C Benavides

Full Text Available Hyperthyroidism during pregnancy is treated with the antithyroid drugs (ATD propylthiouracil (PTU and methimazole (MMI. PTU currently is recommended as the drug of choice during early pregnancy. Yet, despite widespread ATD use in pregnancy, formal studies of ATD teratogenic effects have not been performed.We examined the teratogenic effects of PTU and MMI during embryogenesis in mice. To span different periods of embryogenesis, dams were treated with compounds or vehicle daily from embryonic day (E 7.5 to 9.5 or from E3.5 to E7.5. Embryos were examined for gross malformations at E10.5 or E18.5 followed by histological and micro-CT analysis. Influences of PTU on gene expression levels were examined by RNA microarray analysis.When dams were treated from E7.5 to E9.5 with PTU, neural tube and cardiac abnormalities were observed at E10.5. Cranial neural tube defects were significantly more common among the PTU-exposed embryos than those exposed to MMI or vehicle. Blood in the pericardial sac, which is a feature indicative of abnormal cardiac function and/or abnormal vasculature, was observed more frequently in PTU-treated than MMI-treated or vehicle-treated embryos. Following PTU treatment, a total of 134 differentially expressed genes were identified. Disrupted genetic pathways were those associated with cytoskeleton remodeling and keratin filaments. At E 18.5, no gross malformations were evident in either ATD group, but the number of viable PTU embryos per dam at E18.5 was significantly lower from those at E10.5, indicating loss of malformed embryos. These data show that PTU exposure during embryogenesis is associated with delayed neural tube closure and cardiac abnormalities. In contrast, we did not observe structural or cardiac defects associated with MMI exposure except at the higher dose. We find that PTU exposure during embryogenesis is associated with fetal loss. These observations suggest that PTU has teratogenic potential.

Effect of ambient light exposure of media and embryos on development and quality of porcine parthenogenetically activated embryos

DEFF Research Database (Denmark)

Li, Rong; Liu, Ying; Callesen, Henrik

2015-01-01

Light exposure is a common stress factor during in vitro handling of oocytes and embryos that originates from both microscope and ambient light. In the current study, the effect of two types of ambient light (daylight and laboratory light) on porcine parthenogenetically activated (PA) embryos...... was tested in two experiments: (1) ambient light on medium subsequently used for embryo in vitro development; and (2) ambient light exposure on activated oocytes before in vitro development. The results from Experiment 1 showed that exposure of culture medium to both types of ambient light decreased...... the percentage of blastocysts that showed good morphology, only after 24 h exposure. The results from Experiment 2 revealed a reduction in both blastocyst formation and quality when activated oocytes were exposed to both types of ambient light. This effect was seen after only 1 h exposure and increased with time...

Function of donor cell centrosome in intraspecies and interspecies nuclear transfer embryos

International Nuclear Information System (INIS)

Zhong Zhisheng; Zhang Gang; Meng Xiaoqian; Zhang Yanling; Chen Dayuan; Schatten, Heide; Sun Qingyuan

2005-01-01

Centrosomes, the main microtubule-organizing centers (MTOCs) in most animal cells, are important for many cellular activities such as assembly of the mitotic spindle, establishment of cell polarity, and cell movement. In nuclear transfer (NT), MTOCs that are located at the poles of the meiotic spindle are removed from the recipient oocyte, while the centrosome of the donor cell is introduced. We used mouse MII oocytes as recipients, mouse fibroblasts, rat fibroblasts, or pig granulosa cells as donor cells to construct intraspecies and interspecies nuclear transfer embryos in order to observe centrosome dynamics and functions. Three antibodies against centrin, γ-tubulin, and NuMA, respectively, were used to stain the centrosome. Centrin was not detected either at the poles of transient spindles or at the poles of first mitotic spindles. γ-tubulin translocated into the two poles of the transient spindles, while no accumulated γ-tubulin aggregates were detected in the area adjacent to the two pseudo-pronuclei. At first mitotic metaphase, γ-tubulin was translocated to the spindle poles. The distribution of γ-tubulin was similar in mouse intraspecies and rat-mouse interspecies embryos. The NuMA antibody that we used can recognize porcine but not murine NuMA protein, so it was used to trace the NuMA protein of donor cell in reconstructed embryos. In the pig-mouse interspecies reconstructed embryos, NuMA concentrated between the disarrayed chromosomes soon after activation and translocated to the transient spindle poles. NuMA then immigrated into pseudo-pronuclei. After pseudo-pronuclear envelope breakdown, NuMA was located between the chromosomes and then translocated to the spindle poles of first mitotic metaphase. γ-tubulin antibody microinjection resulted in spindle disorganization and retardation of the first cell division. NuMA antibody microinjection also resulted in spindle disorganization. Our findings indicate that (1) the donor cell centrosome, defined as

Dose-response analysis of phthalate effects on gene expression in rat whole embryo culture

NARCIS (Netherlands)

Robinson, J.F.; Verhoef, A.; van Beelen, V.A.; Pennings, J.L.A.; Piersma, A.H.|info:eu-repo/dai/nl/071276947

2012-01-01

The rat postimplantation whole embryo culture (WEC) model serves as a potential screening tool for developmental toxicity. In this model, cultured rat embryos are exposed during early embryogenesis and evaluated for morphological effects. The integration of molecular-based markers may lead to

Dose-dependent micronuclei formation in normal human fibroblasts exposed to proton radiation

Czech Academy of Sciences Publication Activity Database

Litvinchuk, Alexandra; Vachelová, Jana; Michaelidesová, Anna; Wagner, Richard; Davídková, Marie

2015-01-01

Roč. 54, č. 3 (2015), s. 327-334 ISSN 0301-634X R&D Projects: GA ČR(CZ) GBP108/12/G108; GA MŠk LM2011019 Institutional support: RVO:61389005 Keywords : human fibroblasts * proton radiation * micronuclei assay * biodosimetry Subject RIV: BO - Biophysics Impact factor: 1.923, year: 2015

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

Energy Technology Data Exchange (ETDEWEB)

Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Brandhof, Evert-Jan van den [Center for Environmental Quality, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Pennings, Jeroen L.A. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Kamstra, Jorke H. [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Jelinek, Jaroslav [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Issa, Jean-Pierre J. [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Legler, Juliette [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Ven, Leo T.M. van der, E-mail: leo.van.der.ven@rivm.nl [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands)

2016-01-15

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome

Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

International Nuclear Information System (INIS)

Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M.; Brandhof, Evert-Jan van den; Pennings, Jeroen L.A.; Kamstra, Jorke H.; Jelinek, Jaroslav; Issa, Jean-Pierre J.; Legler, Juliette; Ven, Leo T.M. van der

2016-01-01

Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

International Nuclear Information System (INIS)

Ma, Jane; Bishoff, Bridget; Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane; Castranova, Vincent

2017-01-01

The emission of cerium oxide nanoparticles (CeO 2 ) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO 2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO 2 -induced fibrosis. Male Sprague-Dawley rats were exposed to CeO 2 (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO 2 (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO 2 -exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO 2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO 2 -exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO 2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO 2 nanoparticle exposure. - Highlights: • CeO 2 exposure induced lung fibrosis. • CeO 2 were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO 2 caused ATII cell hypertrophy and hyperplasia and altered fibroblast function

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

Energy Technology Data Exchange (ETDEWEB)

Ma, Jane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Bishoff, Bridget [Mylan Pharmaceuticals, Morganntown, WV (United States); Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Castranova, Vincent, E-mail: vcastran@hsc.wvu.edu [School of Pharmacy, West Virginia University, Morgantown, WV (United States)

2017-05-15

The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII

Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

OpenAIRE

Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

2012-01-01

Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chroma...

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression

DEFF Research Database (Denmark)

Gagliardi, Mark; Maynard, Scott; Miyake, Tetsuaki

2003-01-01

in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF......Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C....../EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had...

Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

Science.gov (United States)

Inoue, Kimiko; Ogura, Atsuo

2013-01-01

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

Study of radio-restoration by various mineral salts and silice wich products (Tuf and Pouzzolane) of rice embryos (oryza-sativa L, Cigalon variety) exposed to cobalt 60 gamma rays

International Nuclear Information System (INIS)

Mouthia-Khadija.

1981-12-01

Mutagenic treatments produce a large number of mutants per unit time and are used for plant improvement. However these treatments cause damage to cells. To counteract this radio-induced damage 2 methods are being tried: - Protection, which consists in the supply of an active product before application of mutagenic agents; - Restoration, which tries to repair the damage after mutagenic treatment. This work is devoted to restoration processes. Technique for the isolation and culture on a suitable nutrient of rice embryos (oryza sativa L, Cigalon variety) separated from non-irradiated caryopses were developed first. By separating out the embryo in this way it is possible to study in vitro the interactions between the embryo and the rest of the caryopsis (albumin + pericarp). The effects of radiations on embryos from caryopses exposed to cobalt 60 gamma rays were measured next, then the action of certain inorganic elements contained in the caryopsis tissues was analysed. On the basis of the first results obtained the differences in response between plantlets from embryos irradiated or otherwise treated or not either by zinc sulphate or by very silice-rich volcanic products (Tuf and Pouzzolane), were examined by chemical analysis techniques. These tests have allowed the detection of ionic changes induced by irradiation during the different stages of plant development and led to a better estimate of the radio-restoration mechanisms brought about by the various chemical compounds used [fr

Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

Science.gov (United States)

Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

2015-11-01

Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

DEFF Research Database (Denmark)

Fusi, Elenora; Rebucci, Raffaella; Pecorini, Chiara

2010-01-01

The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity....... Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P tocopherol...

Efficacy of the Frame and Hu mathematical model for the quantitative analysis of agents influencing growth of chick embryo fibroblasts

International Nuclear Information System (INIS)

Korohoda, K.; Czyz, J.

1994-01-01

The experiments on the effect of various sera and substratum surface area upon growth of chick embryo fibroblasts-like in secondary cultures are described and discussed on the grounds of a mathematical model for growth of anchorage-dependent cells proposed by Frame and Hu. The model and presented results demonstrate the mutual independence of the effects of agent influencing of rate of cell proliferation (i.e. accelerating or retarding growth) and the agents that modify the limitation of cell proliferation (i.e. maximum cell density at confluence). The model proposed by Frame and Hu due to its relative simplicity offers and easy mode of description and quantitative evaluation of experiments concerning cell growth regulation. It is shown that various sera added at constant concentration significantly modify the rate of cell proliferation with little effect upon the maximum cell density attainable. The cells grew much more slowly in the presence of calf serum than in the presence of chick serum and the addition of iron and zinc complexes to calf serum significantly accelerated cell growth. An increase in the substratum surface area by the addition of glass wool to culture vessels significantly increased cell density per constant volume of medium even when retardation of growth was observed. The results presented point to the need of direct cell counting for estimation of cell growth curves and discussion of effects of agents influencing parameters characterizing cell proliferation. (author). 34 refs, 5 figs, 2 tabs

Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma.

Science.gov (United States)

Hanif, Zahid; Ahmed, Farrukh R; Shin, Seung Won; Kim, Young-Kee; Um, Soong Ho

2014-07-01

A controlled preparation of cellulose nanocrystals of different sizes and shapes has been carried out by acid hydrolysis of microcrystalline cellulose. The size- and concentration-dependent toxicity effects of the resulting cellulose nanocrystals were evaluated against two different cell lines, NIH3T3 murine embryo fibroblasts and HCT116 colon adenocarcinoma. It could serve as a therapeutic platform for cancer treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA damage in cultured human skin fibroblasts exposed to excimer laser radiation

Energy Technology Data Exchange (ETDEWEB)

Rimoldi, D.; Miller, A.C.; Freeman, S.E.; Samid, D. (Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD (USA))

1991-06-01

Ultraviolet excimer lasers are being considered for use in a variety of refractive and therapeutic procedures, the long-term biologic consequences of which are unknown. The effect of sublethal doses of 193-nm laser radiation on cellular DNA was examined in cultured human skin fibroblasts. In contrast to 248 nm, treatments with the 193-nm laser radiation below 70 J/m2 did not cause significant pyrimidine dimer formation in the skin cells. This was indicated by the lack of excision repair activities (unscheduled DNA synthesis assay), and further demonstrated by direct analysis of pyrimidine dimers in DNA from irradiated cells. However, a low level of unscheduled DNA synthesis could be detected following irradiation at 193 nm with 70 J/m2. Both the 193-nm and 248-nm radiation were able to induce chromosomal aberrations, as indicated by a micronucleus assay. A dose-dependent increase in micronuclei frequency was observed 48 and 72 h after laser irradiation. These results indicate that exposure of actively replicating human skin fibroblasts to sublethal doses of either 193- or 248-nm laser radiation can result in genotoxicity.

Editor's Highlight: Hydroxyurea Exposure Activates the P53 Signaling Pathway in Murine Organogenesis-Stage Embryos.

Science.gov (United States)

El Husseini, Nazem; Schlisser, Ava E; Hales, Barbara F

2016-08-01

Hydroxyurea, an anticancer agent and potent teratogen, induces oxidative stress and activates a DNA damage response pathway in the gestation day (GD) 9 mouse embryo. To delineate the stress response pathways activated by this drug, we investigated the effect of hydroxyurea exposure on the transcriptome of GD 9 embryos. Timed pregnant CD-1 mice were treated with saline or hydroxyurea (400 mg/kg or 600 mg/kg) on GD 9; embryonic gene and protein expression were examined 3 h later. Microarray analysis revealed that the expression of 1346 probe sets changed significantly in embryos exposed to hydroxyurea compared with controls; the P53 signaling pathway was highly affected. In addition, P53 related family members, P63 and P73, were predicted to be activated and had common and unique downstream targets. Western blot analysis revealed that active phospho-P53 was significantly increased in drug-exposed embryos; confocal microscopy showed that the translocation of phospho-P53 to the nucleus was widespread in the embryo. Furthermore, qRT-PCR showed that the expression of P53-regulated genes (Cdkn1A, Fas, and Trp53inp1) was significantly upregulated in hydroxyurea-exposed embryos; the concentration of the redox sensitive P53INP1 protein was also increased in a hydroxyurea dose-dependent fashion. Thus, hydroxyurea elicits a significant effect on the transcriptome of the organogenesis stage murine embryo, activating several key developmental signaling pathways related to DNA damage and oxidative stress. We propose that the P53 pathway plays a central role in the embryonic stress response and the developmental outcome after teratogen exposure. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Fish embryos on land: terrestrial embryo deposition lowers oxygen uptake without altering growth or survival in the amphibious fish Kryptolebias marmoratus.

Science.gov (United States)

Wells, Michael W; Turko, Andy J; Wright, Patricia A

2015-10-01

Few teleost fishes incubate embryos out of water, but the oxygen-rich terrestrial environment could provide advantages for early growth and development. We tested the hypothesis that embryonic oxygen uptake is limited in aquatic environments relative to air using the self-fertilizing amphibious mangrove rivulus, Kryptolebias marmoratus, which typically inhabits hypoxic, water-filled crab burrows. We found that adult mangrove rivulus released twice as many embryos in terrestrial versus aquatic environments and that air-reared embryos had accelerated developmental rates. Surprisingly, air-reared embryos consumed 44% less oxygen and possessed larger yolk reserves, but attained the same mass, length and chorion thickness. Water-reared embryos moved their opercula ∼2.5 more times per minute compared with air-reared embryos at 7 days post-release, which probably contributed to the higher rates of oxygen uptake and yolk utilization we observed. Genetically identical air- and water-reared embryos from the same parent were raised to maturity, but the embryonic environment did not affect growth, reproduction or emersion ability in adults. Therefore, although aspects of early development were plastic, these early differences were not sustained into adulthood. Kryptolebias marmoratus embryos hatched out of water when exposed to aerial hypoxia. We conclude that exposure to a terrestrial environment reduces the energetic costs of development partly by reducing the necessity of embryonic movements to dispel stagnant boundary layers. Terrestrial incubation of young would be especially beneficial to amphibious fishes that occupy aquatic habitats of poor water quality, assuming low terrestrial predation and desiccation risks. © 2015. Published by The Company of Biologists Ltd.

Use of infrared imaging to predict the developmental stages and differences in chicken embryos exposed to different photoperiods

Science.gov (United States)

Frederick, Rebecca A.; Hsieh, Sheng-Jen; Palomares, Benjamin Giron

2012-06-01

Monitoring development of chicken embryos allows determination of when an egg is not developing and when eggs are close to hatching for more efficient production. Research has been conducted on the effects of temperature fluctuations and light exposure on embryo development; similarities between chicken and mammal embryos; and the use of MRI, tomography, and ultrasound to view specific areas and processes within the embryo. However, there has been little exploration of the use of infrared imaging as a non-destructive method for analyzing and predicting embryonic development. In this study, we built an automated loading system for image acquisition. Pilot experiments were conducted to determine the overall scanning time and scanning frequency. A batch of fertilized eggs was scanned each day as the embryos continued to grow. The captured images were analyzed and categorized into three stages: Stage 1 (days 1 to 7), Stage 2 (days 8 to 14), and Stage 3 (days 15 to 21). The temperature data abstracted from the captured images were divided into two groups. Group 1, consisting of two-thirds of the data, was used to construct a model. Group 2, consisting of one-third of the data, was used to evaluate the predictive accuracy of the model. A three-layer artificial neural network model was developed to predict embryo development stage given a temperature profile. Results suggest that the neural network model is sufficient to predict embryo development stage with good accuracy of 75%. Accuracy can likely be improved if more data sets for each development stage are available.

Dysplasia of the temporo-maxillary joint of mouse embryos due to x-ray irradiation

International Nuclear Information System (INIS)

Shibata, Shigeo

1974-01-01

On the 9-13 pregnant days of ddN mice 200-300 R of X-ray was daily irradiated, and observations were made on the formation of micro-mandible and changes in the temporo-maxillary joint in the embryos on the 18th pregnant day using skeletal and H-E stain specimens. Gross observation revealed the emergence of observable micro-mandible in groups exposed to 200 R on the 10th and 11th pregnant days and in groups exposed to 300 R on the 11th and 12th pregnant days. By skeletal specimens also, micro-mandible was observed in groups exposed on and after the 10th pregnant day, and anomaly of the malar arch was frequently associated with anomaly of the mandibular branches and growth inhibition of the anterior region of the mandible. Histologically, there were observed embryos totally lacking the temporo-maxillary joint composing elements, resulting in fusion with the temporal bone, or embryos lacking the elements partially or complicated by anomaly of cartilagious tissue of the mandibular head. (Mukohata, S.)

Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

Science.gov (United States)

Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

2009-01-01

Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

Developing Xenopus Embryos Recover by Compacting and Expelling Single-Wall Carbon Nanotubes

Science.gov (United States)

Holt, Brian D.; Shawky, Joseph H.; Dahl, Kris Noel; Davidson, Lance A.; Islam, Mohammad F.

2015-01-01

Single-wall carbon nanotubes are high aspect ratio nanomaterials that are being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties, and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single-wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 μm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one-to-two cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call “boluses”. Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. PMID:26153061

Developing Xenopus embryos recover by compacting and expelling single wall carbon nanotubes.

Science.gov (United States)

Holt, Brian D; Shawky, Joseph H; Dahl, Kris Noel; Davidson, Lance A; Islam, Mohammad F

2016-04-01

Single wall carbon nanotubes are high aspect ratio nanomaterials being developed for use in materials, technological and biological applications due to their high mechanical stiffness, optical properties and chemical inertness. Because of their prevalence, it is inevitable that biological systems will be exposed to nanotubes, yet studies of the effects of nanotubes on developing embryos have been inconclusive and are lacking for single wall carbon nanotubes exposed to the widely studied model organism Xenopus laevis (African clawed frog). Microinjection of experimental substances into the Xenopus embryo is a standard technique for toxicology studies and cellular lineage tracing. Here we report the surprising finding that superficial (12.5 ± 7.5 µm below the membrane) microinjection of nanotubes dispersed with Pluronic F127 into one- to two-cell Xenopus embryos resulted in the formation and expulsion of compacted, nanotube-filled, punctate masses, at the blastula to mid-gastrula developmental stages, which we call "boluses." Such expulsion of microinjected materials by Xenopus embryos has not been reported before and is dramatically different from the typical distribution of the materials throughout the progeny of the microinjected cells. Previous studies of microinjections of nanomaterials such as nanodiamonds, quantum dots or spherical nanoparticles report that nanomaterials often induce toxicity and remain localized within the embryos. In contrast, our results demonstrate an active recovery pathway for embryos after exposure to Pluronic F127-coated nanotubes, which we speculate is due to a combined effect of the membrane activity of the dispersing agent, Pluronic F127, and the large aspect ratio of nanotubes. Copyright © 2015 John Wiley & Sons, Ltd.

Embryo splitting

Directory of Open Access Journals (Sweden)

Karl Illmensee

2010-04-01

Full Text Available Mammalian embryo splitting has successfully been established in farm animals. Embryo splitting is safely and efficiently used for assisted reproduction in several livestock species. In the mouse, efficient embryo splitting as well as single blastomere cloning have been developed in this animal system. In nonhuman primates embryo splitting has resulted in several pregnancies. Human embryo splitting has been reported recently. Microsurgical embryo splitting under Institutional Review Board approval has been carried out to determine its efficiency for blastocyst development. Embryo splitting at the 6–8 cell stage provided a much higher developmental efficiency compared to splitting at the 2–5 cell stage. Embryo splitting may be advantageous for providing additional embryos to be cryopreserved and for patients with low response to hormonal stimulation in assisted reproduction programs. Social and ethical issues concerning embryo splitting are included regarding ethics committee guidelines. Prognostic perspectives are presented for human embryo splitting in reproductive medicine.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

International Nuclear Information System (INIS)

Chen, Yuanhong; Huang, Changjiang; Bai, Chenglian; Du, Changchun; Liao, Junhua; Dong, Qiaoxiang

2016-01-01

Highlights: • We developed an in vivo DNA mismatch repair (MMR) measurement assay in zebrafish embryos. • This assay involves microinjection of homo- and heteroduplex EGFP plasmids into zebrafish embryos. • This novel assay was validated with embryos from the MMR-deficient mlh1 mutant fish. • We successfully applied this assay for detecting environmental chemicals with carcinogenic effect. • This novel assay can be used for screening of environmental carcinogens. - Abstract: Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl_2), benzo[a]pyrene (BaP), and

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

Energy Technology Data Exchange (ETDEWEB)

Chen, Yuanhong [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Huang, Changjiang, E-mail: cjhuang5711@163.com [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Bai, Chenglian; Du, Changchun; Liao, Junhua [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Dong, Qiaoxiang, E-mail: dqxdong@163.com [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035 (China)

2016-01-25

Highlights: • We developed an in vivo DNA mismatch repair (MMR) measurement assay in zebrafish embryos. • This assay involves microinjection of homo- and heteroduplex EGFP plasmids into zebrafish embryos. • This novel assay was validated with embryos from the MMR-deficient mlh1 mutant fish. • We successfully applied this assay for detecting environmental chemicals with carcinogenic effect. • This novel assay can be used for screening of environmental carcinogens. - Abstract: Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl{sub 2}), benzo[a]pyrene (BaP), and

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos.

Science.gov (United States)

Manso, Pedro Paulo de Abreu; Dias de Oliveira, Barbara C E P; de Sequeira, Patrícia Carvalho; Maia de Souza, Yuli Rodrigues; Ferro, Jessica Maria dos Santos; da Silva, Igor José; Caputo, Luzia Fátima Gonçalves; Guedes, Priscila Tavares; dos Santos, Alexandre Araujo Cunha; Freire, Marcos da Silva; Bonaldo, Myrna Cristina; Pelajo-Machado, Marcelo

2015-01-01

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos

Science.gov (United States)

Manso, Pedro Paulo de Abreu; Dias de Oliveira, Barbara C. E. P.; de Sequeira, Patrícia Carvalho; Maia de Souza, Yuli Rodrigues; Ferro, Jessica Maria dos Santos; da Silva, Igor José; Caputo, Luzia Fátima Gonçalves; Guedes, Priscila Tavares; dos Santos, Alexandre Araujo Cunha; Freire, Marcos da Silva; Bonaldo, Myrna Cristina; Pelajo-Machado, Marcelo

2015-01-01

The yellow fever (YF) 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system. PMID:26371874

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke-induced alterations

Directory of Open Access Journals (Sweden)

James Elliot Scott

2016-03-01

Full Text Available Background Cigarette smoking is the leading cause of preventable death in the world. It has been implicated in the pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is clearly a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity is the first site of exposure to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases, are proteases which belong to the family of cysteine aspartic acid proteases and are the key components for the downstream amplification of intra-cellular apoptotic signals. Of the 14 caspases known, caspase-3 is the key executioner of apoptosis. Fetal rat lung fibroblasts but not PDL viability is reduced by exposure to CSE. In addition Caspase 3 activity is elevated after CSE exposure in fetal lung fibroblasts but not in PDLs. Expression of caspase 3 is induced in CSE exposed lung fibroblasts but not in PDLs. Caspase 3 was localized to the cytoplasm in both cell types.

Polycyclic aromatic hydrocarbons disrupt axial development in sea urchin embryos through a β-catenin dependent pathway

International Nuclear Information System (INIS)

Pillai, Murali C.; Vines, Carol A.; Wikramanayake, Athula H.; Cherr, Gary

2003-01-01

Sea urchin (Lytechinus anemesis) embryos were used as an experimental system to investigate the mechanisms of the developmental toxicity of creosote, one of the most widely used wood preserving chemicals, as well as some of its polycyclic aromatic hydrocarbon (PAH) constituents (phenanthrene, fluoranthene, fluorene, pyrene and quinoline). Data suggest that creosote and PAHs affect axial development and patterning in sea urchin embryos by disrupting the regulation of β-catenin, a crucial transcriptional co-activator of specific target genes in the Wnt/wg signaling pathway. When ciliated blastula stage embryos were exposed to these compounds, they developed into exogastrulae with completely evaginated archentera, demonstrating that these chemicals disrupt axial development and patterning. This response occurred in a dose-dependent fashion, with the EC 50 of creosote for complete exogastrulation being 1.57 ppm, while the EC 50 s of the PAHs ranged from 0.41 ppm (2.0 μM) to 4.33 ppm (33.5 μM). Morphologically, the exogastrulae that developed from embryos exposed to creosote and PAHs appeared to be identical to those that resulted from exposure to lithium chloride, a classical agent known to induce vegetalization and exogastrulation in sea urchin embryos. Immunological studies using antibodies against β-catenin, a multi-functional protein known to be involved in cell-cell adhesion and cell fate specification during embryonic development, revealed high levels of nuclear accumulation of β-catenin by cells of creosote- and PAH-exposed embryos, irrespective of their positions in the developing embryo. Dissociated embryonic cells cultured in the presence of these agents rapidly responded in a similar fashion. Since β-catenin accumulation occurs in nuclei of several types of cancer cells, it is possible this may be a general mechanism by which PAHs affect a variety of different cell types

Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains

Energy Technology Data Exchange (ETDEWEB)

Paffenholz, V.

1978-02-01

Primary mouse fibroblast cultures were established from 10 day old embryos of 3 inbred strains with a genetically determined different life expectancy. The capacity for unscheduled DNA synthesis following uv irradiation was studied in these cells at various passage levels of the in vitro ageing process. The mouse fibroblasts show considerable repair synthesis corresponding to the duration of exposure time. The capacity for induction of unscheduled DNA synthesis was different in the cells of each strain and correlated to the natural life span of the animal. In each case, however, the ability to perform repair synthesis was subjected to an age-associated decline, although semiconservative DNA synthesis and proliferative potential of the cell was not changed until the cultures entered phase III passages.

Acrolein-Exposed Normal Human Lung Fibroblasts in Vitro: Cellular Senescence, Enhanced Telomere Erosion, and Degradation of Werner’s Syndrome Protein

Science.gov (United States)

Jang, Jun-Ho; Bruse, Shannon; Huneidi, Salam; Schrader, Ronald M.; Monick, Martha M.; Lin, Yong; Carter, A. Brent; Klingelhutz, Aloysius J.

2014-01-01

Background: Acrolein is a ubiquitous environmental hazard to human health. Acrolein has been reported to activate the DNA damage response and induce apoptosis. However, little is known about the effects of acrolein on cellular senescence. Objectives: We examined whether acrolein induces cellular senescence in cultured normal human lung fibroblasts (NHLF). Methods: We cultured NHLF in the presence or absence of acrolein and determined the effects of acrolein on cell proliferative capacity, senescence-associated β-galactosidase activity, the known senescence-inducing pathways (e.g., p53, p21), and telomere length. Results: We found that acrolein induced cellular senescence by increasing both p53 and p21. The knockdown of p53 mediated by small interfering RNA (siRNA) attenuated acrolein-induced cellular senescence. Acrolein decreased Werner’s syndrome protein (WRN), a member of the RecQ helicase family involved in DNA repair and telomere maintenance. Acrolein-induced down-regulation of WRN protein was rescued by p53 knockdown or proteasome inhibition. Finally, we found that acrolein accelerated p53-mediated telomere shortening. Conclusions: These results suggest that acrolein induces p53-mediated cellular senescence accompanied by enhanced telomere attrition and WRN protein down-regulation. Citation: Jang JH, Bruse S, Huneidi S, Schrader RM, Monick MM, Lin Y, Carter AB, Klingelhutz AJ, Nyunoya T. 2014. Acrolein-exposed normal human lung fibroblasts in vitro: cellular senescence, enhanced telomere erosion, and degradation of Werner’s syndrome protein. Environ Health Perspect 122:955–962; http://dx.doi.org/10.1289/ehp.1306911 PMID:24747221

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences.

Directory of Open Access Journals (Sweden)

Andrei Tintu

Full Text Available Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos.Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20 and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF and its scavenger soluble VEGF receptor-1 (sFlt-1 to investigate the potential role of this hypoxia-regulated cytokine.Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype.Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Chloride transport in human fibroblasts is activated by hypotonic shock

Energy Technology Data Exchange (ETDEWEB)

Rugolo, M.; Mastocola, T.; Flamigni, A.; Lenaz, G. (Universita' di Bologna (Italy))

1989-05-15

Incubation of human skin fibroblasts in hypotonic media induced the activation of {sup 36}Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of {sup 36}Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also {sup 36}Cl- influx was enhanced by hypotonic medium.

Disentangling the multifactorial contributions of fibronectin, collagen and cyclic strain on MMP expression and extracellular matrix remodeling by fibroblasts

NARCIS (Netherlands)

Zhang, Y.; Lin, Z.; Foolen, J.; Schoen, I.; Santoro, A.; Zenobi-Wong, M.; Vogel, Viola

2014-01-01

Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was

Effects of temperature on gene expression in embryos of the coral Montastraea faveolata.

KAUST Repository

Voolstra, Christian R.; Schnetzer, Julia; Peshkin, Leonid; Randall, Carly J; Szmant, Alina M; Medina, Mó nica

2009-01-01

Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5 degrees C, 29.0 degrees C, and 31.5 degrees C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours.

Effects of temperature on gene expression in embryos of the coral Montastraea faveolata.

KAUST Repository

Voolstra, Christian R.

2009-12-23

Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5 degrees C, 29.0 degrees C, and 31.5 degrees C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours.

Time to take human embryo culture seriously.

Science.gov (United States)

Sunde, Arne; Brison, Daniel; Dumoulin, John; Harper, Joyce; Lundin, Kersti; Magli, M Cristina; Van den Abbeel, Etienne; Veiga, Anna

2016-10-01

Is it important that end-users know the composition of human embryo culture media? We argue that there is as strong case for full transparency concerning the composition of embryo culture media intended for human use. Published data suggest that the composition of embryo culture media may influence the phenotype of the offspring. A review of the literature was carried out. Data concerning the potential effects on embryo development of culture media were assessed and recommendations for users made. The safety of ART procedures, especially with respect to the health of the offspring, is of major importance. There are reports from the literature indicating a possible effect of culture conditions, including culture media, on embryo and fetal development. Since the introduction of commercially available culture media, there has been a rapid development of different formulations, often not fully documented, disclosed or justified. There is now evidence that the environment the early embryo is exposed to can cause reprogramming of embryonic growth leading to alterations in fetal growth trajectory, birthweight, childhood growth and long-term disease including Type II diabetes and cardiovascular problems. The mechanism for this is likely to be epigenetic changes during the preimplantation period of development. In the present paper the ESHRE working group on culture media summarizes the present knowledge of potential effects on embryo development related to culture media, and makes recommendations. There is still a need for large prospective randomized trials to further elucidate the link between the composition of embryo culture media used and the phenotype of the offspring. We do not presently know if the phenotypic changes induced by in vitro embryo culture represent a problem for long-term health of the offspring. Published data indicate that there is a strong case for demanding full transparency concerning the compositions of and the scientific rationale behind the

Hormetic effect induced by depleted uranium in zebrafish embryos

International Nuclear Information System (INIS)

Ng, C.Y.P.; Cheng, S.H.; Yu, K.N.

2016-01-01

Highlights: • Studied hormetic effect induced by uranium (U) in embryos of zebrafish (Danio rerio). • Hormesis observed at 24 hpf for exposures to 10 μg/l of depleted U (DU). • Hormesis not observed before 30 hpf for exposures to 100 μg/l of DU. • Hormetic effect induced in zebrafish embryos in a dose-and time-dependent manner. - Abstract: The present work studied the hormetic effect induced by uranium (U) in embryos of zebrafish (Danio rerio) using apoptosis as the biological endpoint. Hormetic effect is characterized by biphasic dose-response relationships showing a low-dose stimulation and a high-dose inhibition. Embryos were dechorionated at 4 h post fertilization (hpf), and were then exposed to 10 or 100 μg/l depleted uranium (DU) in uranyl acetate solutions from 5 to 6 hpf. For exposures to 10 μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20 hpf but were significantly decreased at 24 hpf, which demonstrated the presence of U-induced hormesis. For exposures to 100 μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20, 24 and 30 hpf. Hormetic effect was not shown but its occurrence between 30 and 48 hpf could not be ruled out. In conclusion, hormetic effect could be induced in zebrafish embryos in a concentration- and time-dependent manner.

Hormetic effect induced by depleted uranium in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Ng, C.Y.P. [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); Cheng, S.H., E-mail: bhcheng@cityu.edu.hk [Department of Biomedical Sciences, City University of Hong Kong (Hong Kong); State Key Laboratory in Marine Pollution, City University of Hong Kong (Hong Kong); Yu, K.N., E-mail: peter.yu@cityu.edu.hk [Department of Physics and Materials Science, City University of Hong Kong (Hong Kong); State Key Laboratory in Marine Pollution, City University of Hong Kong (Hong Kong)

2016-06-15

Highlights: • Studied hormetic effect induced by uranium (U) in embryos of zebrafish (Danio rerio). • Hormesis observed at 24 hpf for exposures to 10 μg/l of depleted U (DU). • Hormesis not observed before 30 hpf for exposures to 100 μg/l of DU. • Hormetic effect induced in zebrafish embryos in a dose-and time-dependent manner. - Abstract: The present work studied the hormetic effect induced by uranium (U) in embryos of zebrafish (Danio rerio) using apoptosis as the biological endpoint. Hormetic effect is characterized by biphasic dose-response relationships showing a low-dose stimulation and a high-dose inhibition. Embryos were dechorionated at 4 h post fertilization (hpf), and were then exposed to 10 or 100 μg/l depleted uranium (DU) in uranyl acetate solutions from 5 to 6 hpf. For exposures to 10 μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20 hpf but were significantly decreased at 24 hpf, which demonstrated the presence of U-induced hormesis. For exposures to 100 μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20, 24 and 30 hpf. Hormetic effect was not shown but its occurrence between 30 and 48 hpf could not be ruled out. In conclusion, hormetic effect could be induced in zebrafish embryos in a concentration- and time-dependent manner.

Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

Energy Technology Data Exchange (ETDEWEB)

Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

2004-07-14

Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

Piglets produced by transfer of vitrified porcine embryos after stepwise dilution of cryoprotectants.

Science.gov (United States)

Kobayashi, S; Takei, M; Kano, M; Tomita, M; Leibo, S P

1998-02-01

A total of 498 porcine embryos at various stages of development collected from superovulated gilts was used to investigate cryopreservation. First, blastocysts (BL), expanded blastocysts (ExB), and hatched blastocysts (HB) were used to determine the effect of exposure to concentrated solutions of ethylene glycol as cryoprotective additives (CPAs) on embryo survival. Then, survival of other embryos after vitrification by rapid cooling was determined. Based on their development after 48 h in culture, embryos were not injured by being exposed to 2.0 M ethylene glycol (EG) for 15 min or to 2.0 M EG for 5 min and then to a solution of 8.0 M EG in 7% polyvinylpyrrolidone (PVP) for 1 min. The CPAs were removed from the embryos by diluting them with 1.7 M galactose. To vitrify the embryos, they were exposed to 2.0 M EG for 5 min and then were pipetted directly into short columns of 8.0 M EG-PVP contained within (1.25-ml plastic straws and separated from long columns of 1.7 M galactose by an air bubble. The straws were plunged directly into LN2. After the straws were warmed rapidly in a 25 degrees C water bath, the embryos were immediately mixed with galactose within the straws by shaking them vigorously to mix the contents. In sequential experiments, three methods were used to dilute the CPA solutions. Method 1: Embryos in the EG-PVP-galactose mixture were expelled from the straws and rinsed and cultured in modified CZB medium (mCZB). Method II: Embryos in the mixture were placed briefly into 1.5 M EG and then rinsed and cultured in mCZB. Method III: Embryos in the mixture were rinsed in 1.0 M EG and then in 0.5 M EG and finally rinsed with mCZB and cultured. After 48 h in culture, the respective percentages of survival of embryos vitrified as BL, ExB, or HB were: Method I, 21, 32, and 13%; Method II, 9, 40, and 24%; Method III, 35, 85, and 71%. Of 20 additional ExB vitrified embryos diluted by Method III and transferred into a recipient, four developed into live piglets

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture.

Science.gov (United States)

Miller-Pinsler, Lutfiya; Wells, Peter G

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat(b)/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug=GD 1), exposed for 24h to 2 or 4mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (pcatalase (PEG-cat) 8h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (pcatalase is a determinant of risk for EtOH embryopathies. Copyright © 2015 Elsevier Inc. All rights reserved.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

International Nuclear Information System (INIS)

Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

2010-01-01

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

Energy Technology Data Exchange (ETDEWEB)

Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

2010-01-05

The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

Viability of bovine demi embryo after splitting of fresh and frozen thawed embryo derived from in vitro embryo production

Directory of Open Access Journals (Sweden)

M Imron

2007-06-01

Full Text Available In vivo embryo production was limited by number of donor, wide variability respond due to superovulation program and also immunoactifity of superovulation hormone (FSH. Splitting technology could be an alternative to increase the number of transferrable embryos into recipien cows. Splitting is done with cutting embryo becoming two equal pieces (called demi embrio base on ICM orientation. The objective of this research was to determine the viability of demi embryo obtained from embryo splitting of fresh and frozen thawed embryo. The results showed that demi embryos which performed blastocoel reexpansion 3 hours after embryo splitting using fresh and frozen thawed embryos were 76.9 and 76.2% respectively. Base on existention of inner cell mass (ICM, the number of demi embryos developed with ICM from fresh and frozen thawed embryos were not significantly different (90.6 and 85.7% respectively. The cell number of demi embryo from fresh embryos splitting was not different compared with those from frozen thawed embryos (36.1 and 35.9 respectively. These finding indicated that embryo splitting can be applied to frozen thawed embryos with certain condition as well as fresh embryos.

Elastin hydrolysate derived from fish enhances proliferation of human skin fibroblasts and elastin synthesis in human skin fibroblasts and improves the skin conditions.

Science.gov (United States)

Shiratsuchi, Eri; Nakaba, Misako; Yamada, Michio

2016-03-30

Recent studies have shown that certain peptides significantly improve skin conditions, such as skin elasticity and the moisture content of the skin of healthy woman. This study aimed to investigate the effects of elastin hydrolysate on human skin. Proliferation and elastin synthesis were evaluated in human skin fibroblasts exposed to elastin hydrolysate and proryl-glycine (Pro-Gly), which is present in human blood after elastin hydrolysate ingestion. We also performed an ingestion test with elastin hydrolysate in humans and evaluated skin condition. Elastin hydrolysate and Pro-Gly enhanced the proliferation of fibroblasts and elastin synthesis. Maximal proliferation response was observed at 25 ng mL(-1) Pro-Gly. Ingestion of elastin hydrolysate improved skin condition, such as elasticity, number of wrinkles, and blood flow. Elasticity improved by 4% in the elastin hydrolysate group compared with 2% in the placebo group. Therefore, elastin hydrolysate activates human skin fibroblasts and has beneficial effects on skin conditions. © 2015 Society of Chemical Industry.

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

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Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

High inorganic phosphate causes DNMT1 phosphorylation and subsequent fibrotic fibroblast activation

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Tan, Xiaoying [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Xu, Xingbo [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); Zeisberg, Elisabeth M. [Department of Cardiology and Pneumology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany); Zeisberg, Michael, E-mail: mzeisberg@med.uni-goettingen.de [Department of Nephrology and Rheumatology, Göttingen University Medical Center, Georg August University, Göttingen (Germany); German Center for Cardiovascular Research (DZHK), Göttingen (Germany)

2016-04-08

Phosphate is an essential constituent of critical cellular functions including energy metabolism, nucleic acid synthesis and phosphorylation-dependent cell signaling. Increased plasma phosphate levels are an independent risk factor for lowered life-expectancy as well as for heart and kidney failure. Nevertheless, direct cellular effects of elevated phosphate concentrations within the microenvironment are poorly understood and have been largely neglected in favor of phosphor-regulatory hormones. Because interstitial fibrosis is the common determinant of chronic progressive kidney disease, and because fibroblasts are major mediators of fibrogenesis, we here explored the effect of high extracellular phosphate levels on renal fibroblasts. We demonstrate that high inorganic phosphate directly induces fibrotic fibroblast activation associated with increased proliferative activity, increased expression of α-smooth muscle actin and increased synthesis of type I collagen. We further demonstrate that such fibroblast activation is dependent on phosphate influx, aberrant phosphorylation of DNA methyltransferase DNMT1 and aberrant CpG island promoter methylation. In summary, our studies demonstrate that elevated phosphate concentrations induce pro-fibrotic fibroblast activation independent of phospho-regulatory hormones. - Highlights: • We exposed human kidney fibroblasts to media containing 1 mM or 3 mM phosphate. • Increased phosphate influx causes phosphorylation of DNA methyltransferase Dnmt1. • Phosphorylated Dnmt1 causes promoter methylation and transcriptional silencing of RASAL1. • Depletion of RASAL1 causes increased intrinsic Ras-GTP activity and fibroblast activation. • Inorganic phosphate causes fibroblast activation independent of phospho-regulatory hormones.

Developmental Toxicity Assay for Food Additive Tartrazine Using Zebrafish ( Danio rerio) Embryo Cultures.

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Joshi, Vani; Katti, Pancharatna

Tartrazine (TTZ) is an azo dye used as a colorant in food products, drugs, and cosmetics. The present study evaluates the impacts of TTZ on embryonic development of zebrafish ( Danio rerio). Laboratory-raised D. rerio embryos (n = 20/concentration) were exposed to graded dilutions of TTZ (0, 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, and 100 mM) from gastrulation stage (5.25 hours postfertilization [hpf]) until hatching and developmental trajectory was traced up to day 7. The no observed effect concentration (NOEC), median lethal concentration (LC 50 ), median effective concentration (EC 50 ), and teratogenic index (TI) were calculated. Exposure of embryos to effects; 20 to 30 mM TTZ caused tail bending, cardiac and yolk sac edema in 50% of larvae; in 30 to 50 mM TTZ-exposed embryos the heart rates declined along with the above mentioned deformities, causing mortality within 96 to 144 hpf; development ceased completely at 75 to 100 mM concentration. The NOEC and LC 50 were recorded at 5 and 29.4 mM dose, respectively. The EC 50 values for heart rate, cardiac edema, tail bending, and hatching success were at 59.60, 53.81, 98.28, and 58.97 mM with TI quotient 0.49, 0.54, 0.29, and 0.49, respectively. We conclude that TTZ is not embryo toxic/teratogenic for zebrafish embryos up to a dose level of 10 mM concentration.

Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency.

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Pang, Yun-Wei; An, Lei; Wang, Peng; Yu, Yong; Yin, Qiu-Dan; Wang, Xiao-Hong; Xin-Zhang; Qian-Zhang; Yang, Mei-Ling; Min-Guo; Wu, Zhong-Hong; Tian, Jian-Hui

2013-05-01

This study was conducted to investigate the effect of melatonin during the culture of donor cells and cloned embryos on the in vitro developmental competence and quality of cloned porcine embryos. At concentrations of 10(-6 )M or 10(-8) M, melatonin significantly enhanced the proliferation of porcine fetal fibroblasts (PFFs), and the blastocyst rate was significantly increased in the 10(-10) M melatonin-treated donor cell group. Cloned embryo development was also improved in embryo culture medium that was supplemented with 10(-9) M or 10(-12) M melatonin. When both donor cells and cloned embryos were treated with melatonin, the cleavage rate and total cell number of blastocysts were not significantly affected; however, the blastocyst rate was increased significantly (20.0% versus 11.7%). TUNEL assays showed that combined melatonin treatment reduced the rate of apoptotic nuclei (3.6% versus 6.1%). Gene expression analysis of the apoptosis-related genes BAX, BCL2L1, and p53 showed that the expression of BCL2L1 was significantly elevated 2.7-fold relative to the control group, while the expression of BAX and p53 was significantly decreased by 3.7-fold and 23.2-fold, respectively. In addition, we detected the expression of two melatonin receptors (MT1 and MT2) in PFFs but not in porcine cloned embryos. We conclude that exogenous melatonin enhances the development of porcine cloned embryos and improves embryo quality by inhibiting p53-mediated apoptotic pathway. The proliferation of PFFs may be mediated by receptor binding, but the beneficial effects of melatonin on embryonic development may be receptor-independent, possibly through melatonin's ability to directly scavenge free radicals. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

Cell-free assay measuring repair DNA synthesis in human fibroblasts

International Nuclear Information System (INIS)

Ciarrocchi, G.; Linn, S.

1978-01-01

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of uv-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methylmethanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of uv-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro

Yellow Fever 17DD Vaccine Virus Infection Causes Detectable Changes in Chicken Embryos.

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Pedro Paulo de Abreu Manso

Full Text Available The yellow fever (YF 17D vaccine is one of the most effective human vaccines ever created. The YF vaccine has been produced since 1937 in embryonated chicken eggs inoculated with the YF 17D virus. Yet, little information is available about the infection mechanism of YF 17DD virus in this biological model. To better understand this mechanism, we infected embryos of Gallus gallus domesticus and analyzed their histopathology after 72 hours of YF infection. Some embryos showed few apoptotic bodies in infected tissues, suggesting mild focal infection processes. Confocal and super-resolution microscopic analysis allowed us to identify as targets of viral infection: skeletal muscle cells, cardiomyocytes, nervous system cells, renal tubular epithelium, lung parenchyma, and fibroblasts associated with connective tissue in the perichondrium and dermis. The virus replication was heaviest in muscle tissues. In all of these specimens, RT-PCR methods confirmed the presence of replicative intermediate and genomic YF RNA. This clearer characterization of cell targets in chicken embryos paves the way for future development of a new YF vaccine based on a new cell culture system.

Toxicity of buprofezin on the survival of embryo and larvae of African catfish, Clarias gariepinus (Bloch).

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Marimuthu, Kasi; Muthu, Narmataa; Xavier, Rathinam; Arockiaraj, Jesu; Rahman, M Aminur; Subramaniam, Sreeramanan

2013-01-01

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (pbuprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC50 value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (pbuprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC50 values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC50 values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.

Blue light-irradiated human keloid fibroblasts: an in vitro study

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Magni, Giada; Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Coppi, Elisabetta; Cherchi, Federica; Fusco, Irene; Pugliese, Anna Maria; Pedata, Felicita; Fraccalvieri, Marco; Gasperini, Stefano; Pavone, Francesco S.; Tripodi, Cristina; Alfieri, Domenico; Targetti, Lorenzo

2018-02-01

Blue LED light irradiation is currently under investigation because of its effect in wound healing improvement. In this context, several mechanisms of action are likely to occur at the same time, consistently with the presence of different light absorbers within the skin. In our previous studies we observed the wound healing in superficial abrasions in an in vivo murine model. The results evidenced that both inflammatory infiltrate and myofibroblasts activity increase after irradiation. In this study we focused on evaluating the consequences of light absorption in fibroblasts from human cells culture: they play a key role in wound healing, both in physiological conditions and in pathological ones, such as keloid scarring. In particular we used keloids fibroblasts as a new target in order to investigate a possible metabolic or cellular mechanism correlation. Human keloid tissues were excised during standard surgery and immediately underwent primary cell culture extraction. Fibroblasts were allowed to grow in the appropriate conditions and then exposed to blue light. A metabolic colorimetric test (WST-8) was then performed. The tests evidenced an effect in mitochondrial activity, which could be modulated by the duration of the treatment. Electrophysiology pointed out a different behavior of irradiated fibroblasts. In conclusion, the Blue LED light affects the metabolic activity of fibroblasts and thus the cellular proliferation rate. No specific effect was found on keloid fibroblasts, thus indicating a very basic intracellular component, such as cytochromes, being the target of the treatment.

Ultrastructural changes following electron irradiation in three-dimensional culture of normal human dermal fibroblasts

International Nuclear Information System (INIS)

Han, Chunmao; Ishikura, Naotaka; Tsukada, Sadao

1994-01-01

The present study was designed to examine the effect of electron irradiation on fibroblasts and extracellular matrices electron-microscopically. The three-dimensional dermal fibroblast culture was exposed to one, 4 or 10 Gy of electron beams. One day after irradiation, fibroblasts were vacuolated in all irradiated groups and intercellular spaces were increased in a dose-dependent manner. Seven days later, intercellular spaces became dense in both one and 4 Gy groups, although they were still extremely increased in the 10 Gy group. The remaining fibroblasts were still activated in all groups. Thirty days after irradiation, myofibroblastic cells were scarcely observed, but extracellular fine fibrils and collagen fibrils were observed in all irradiated groups. The other ultrastructural findings were similar to those in the control group. In conclusion, electron beams damaged not only cells but also extracellular matrix. The extracellular matrix may be repaired by activated residual fibroblasts, resulting in the mixture of new and old collagen fibrils having different diamters. (N.K.)

No cytotoxic effects from application of pentoxifylline to spermatozoa on subsequent pre-implantation embryo development in mice

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Mohammad Ali Khalili

2017-06-01

Full Text Available The aim was to assess the effect of spermatozoa exposed to PTX on the rates of fertilization and embryo development and apoptotic cells within blastocysts in an animal model. Mice Oocytes were inseminated with spermatozoa exposed to 3.6 mmol PTX for 30 min, or with neat spermatozoa. Then fertilization and embryo development rate, blastocyst formation and quality, as well as total cell number of blastocyst, and DNA fragmentation index (DFI in blastocysts were surveyed in both groups. Fertilization and embryo development rate were similar between the groups. The rates of blastocyst formation did not differ significantly between control and PTX groups (52.4% vs. 51.8%. The average of total cell count in blastocysts and DFI in control and PTX groups were also insignificant (31.08 ± 1.5 vs. 34.14 ± 1.5 and 9.76 ± 5.0 vs. 11.77 ± 5.4. Application of PTX for enhancing sperm motility does not cause a cytotoxic effect on subsequent embryo development and embryo genome integrity.

Embryo density and medium volume effects on early murine embryo development.

Science.gov (United States)

Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

1992-10-01

One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

Toxicity of buprofezin on the survival of embryo and larvae of African catfish, Clarias gariepinus (Bloch.

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Kasi Marimuthu

Full Text Available Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p0.05 in the LC50 values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.

Shorter exposures to harder X-rays trigger early apoptotic events in Xenopus laevis embryos.

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JiaJia Dong

Full Text Available BACKGROUND: A long-standing conventional view of radiation-induced apoptosis is that increased exposure results in augmented apoptosis in a biological system, with a threshold below which radiation doses do not cause any significant increase in cell death. The consequences of this belief impact the extent to which malignant diseases and non-malignant conditions are therapeutically treated and how radiation is used in combination with other therapies. Our research challenges the current dogma of dose-dependent induction of apoptosis and establishes a new parallel paradigm to the photoelectric effect in biological systems. METHODOLOGY/PRINCIPAL FINDINGS: We explored how the energy of individual X-ray photons and exposure time, both factors that determine the total dose, influence the occurrence of cell death in early Xenopus embryo. Three different experimental scenarios were analyzed and morphological and biochemical hallmarks of apoptosis were evaluated. Initially, we examined cell death events in embryos exposed to increasing incident energies when the exposure time was preset. Then, we evaluated the embryo's response when the exposure time was augmented while the energy value remained constant. Lastly, we studied the incidence of apoptosis in embryos exposed to an equal total dose of radiation that resulted from increasing the incoming energy while lowering the exposure time. CONCLUSIONS/SIGNIFICANCE: Overall, our data establish that the energy of the incident photon is a major contributor to the outcome of the biological system. In particular, for embryos exposed under identical conditions and delivered the same absorbed dose of radiation, the response is significantly increased when shorter bursts of more energetic photons are used. These results suggest that biological organisms display properties similar to the photoelectric effect in physical systems and provide new insights into how radiation-mediated apoptosis should be understood and

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

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Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

2018-03-12

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

International Nuclear Information System (INIS)

Miller-Pinsler, Lutfiya; Wells, Peter G.

2015-01-01

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat b /J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • EtOH developmental

Embryonic catalase protects against ethanol embryopathies in acatalasemic mice and transgenic human catalase-expressing mice in embryo culture

Energy Technology Data Exchange (ETDEWEB)

Miller-Pinsler, Lutfiya [Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Wells, Peter G., E-mail: pg.wells@utoronto.ca [Division of Biomolecular Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario (Canada); Department of Pharmacology and Toxicology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada)

2015-09-15

Reactive oxygen species (ROS) have been implicated in the mechanism of ethanol (EtOH) teratogenicity, but the protective role of the embryonic antioxidative enzyme catalase is unclear, as embryonic activity is only about 5% of maternal levels. We addressed this question in a whole embryo culture model. C57BL/6 mouse embryos expressing human catalase (hCat) or their wild-type (C57BL/6 WT) controls, and C3Ga.Cg-Cat{sup b}/J catalase-deficient, acatalasemic (aCat) mouse embryos or their wild-type C3HeB/FeJ (C3H WT) controls, were explanted on gestational day (GD) 9 (plug = GD 1), exposed for 24 h to 2 or 4 mg/mL EtOH or vehicle, and evaluated for functional and morphological changes. hCat and C57BL/6 WT vehicle-exposed embryos developed normally, while EtOH was embryopathic in C57BL/6 WT embryos, evidenced by decreases in anterior neuropore closure, somites developed, turning and head length, whereas hCat embryos were protected (p < 0.001). Maternal pretreatment of C57BL/6 WT dams with 50 kU/kg PEG-catalase (PEG-cat) 8 h prior to embryo culture, which increases embryonic catalase activity, blocked all EtOH embryopathies (p < 0.001). Vehicle-exposed aCat mouse embryos had lower yolk sac diameters compared to WT controls, suggesting that endogenous ROS are embryopathic. EtOH was more embryopathic in aCat embryos than WT controls, evidenced by reduced head length and somite development (p < 0.01), and trends for reduced anterior neuropore closure, turning and crown–rump length. Maternal pretreatment of aCat dams with PEG-Cat blocked all EtOH embryopathies (p < 0.05). These data suggest that embryonic catalase is a determinant of risk for EtOH embryopathies. - Highlights: • Ethanol (EtOH) exposure causes structural embryopathies in embryo culture. • Genetically enhanced catalase (hCat) protects against EtOH embryopathies. • Genetically deficient catalase (aCat) exacerbates EtOH embryopathies. • Embryonic catalase is developmentally important. • Et

Induction of thermal shock proteins and changes in radiosensitivity after heat treatment of Bombyx mori L. embryos

International Nuclear Information System (INIS)

Agaev, F.A.

1993-01-01

The method of gel-electrophoresis was used to study thermal shock protein synthesis in Bombyx mori embryos exposed to a mixture of heat and gamma-radiation. Induction of thermal shock protein synthesis was not inhibited by gamma-radiation. It is suggested that thermal shock proteins are involved embryo radiosensitivity modification

Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

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Anitua, Eduardo; de la Fuente, María; Ferrando, Marcos; Quintana, Fernando; Larreategui, Zaloa; Matorras, Roberto; Orive, Gorka

2016-11-01

To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effect of intrauterine position on the radiosensitivity of rat embryos

International Nuclear Information System (INIS)

Ward, W.F.; Aceto, H. Jr.; Karp, C.H.

1977-01-01

Rats were exposed to gamma rays or helium ions on one of days 4 to 9 of gestation. Embryonic survival was recorded as a function of intrauterine position at autopsy on day 20 of gestation. Embryos located at the ovarian and cervical ends of the uterus experienced higher rates of mortality than did their littermates located at the middle of the uterine horn. This effect was observed in litters exposed to both radiation modalities on all days studied. The influence of intrauterine position on embryonic survival was directly proportional to radiation dose and to the number of fetuses occupying the uterus horn. Under the least advantageous conditions (i.e., a crowded uterine horn exposed to a moderately high radiation dose), the cervical embryo's probability of survival was less than half that of the litter as a whole. A disproportionately high rate of embryonic mortality at the cervical position was also observed in litters irradiated under hypoxic conditions, suggesting that the non-random distribution of radiation effect was not the result of variations in oxygen concentration within the uterus. In contrast, there was no indication that intrauterine position influenced the distribution of gross morphologic abnormalities in irradiated litters

DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

International Nuclear Information System (INIS)

Zhang, Bingzhen; Shen, Chunzi; Yang, Liu; Li, Chunhui; Yi, Anji; Wang, Zhiping

2013-01-01

Carbon disulfide (CS 2 ) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS 2 via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD 50 (157.85 mg/kg), 0.2 LD 50 (315.7 mg/kg) and 0.4 LD 50 (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS 2 . - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss

Pock forming ability of fowl pox virus isolated from layer chicken and its adaptation in chicken embryo fibroblast cell culture.

Science.gov (United States)

Gilhare, Varsha Rani; Hirpurkar, S D; Kumar, Ashish; Naik, Surendra Kumar; Sahu, Tarini

2015-03-01

The objective of the present study was to examine pock forming ability of field strain and vaccine strain of fowl pox virus (FPV) in chorioallantoic membrane (CAM) of embryonated chicken eggs and its adaptation in chicken embryo fibroblast (CEF) cell culture. Dry scabs were collected from 25 affected birds in glycerin-saline and preserved at 4°C until processed. Virus was isolated in 10-day-old embryonated chicken eggs by dropped CAM method. The identity of the virus is confirmed by clinical findings of affected birds, pock morphology and histopathology of infected CAM. In addition one field isolate and vaccine strain of FPV was adapted to CEF cell culture. CEF cell culture was prepared from 9-day-old embryonated chicken eggs. Clinical symptoms observed in affected birds include pox lesion on comb, wattle, eyelids and legs, no internal lesions were observed. All field isolates produced similar findings in CAM. Pocks produced by field isolates ranged from 3 mm to 5 mm at the third passage while initial passages edematous thickening and necrosis of CAM was observed. Pocks formed by lyophilized strain were ranges from 0.5 mm to 2.5 mm in diameter scattered all over the membrane at the first passage. Intra-cytoplasmic inclusion bodies are found on histopathology of CAM. At third passage level, the CEF inoculated with FPV showed characteristic cytopathic effect (CPE) included aggregation of cells, syncytia and plaque formation. FPV field isolates and vaccine strain produced distinct pock lesions on CAMs. Infected CAM showed intracytoplasmic inclusion bodies. The CEF inoculated with FPV field isolate as well as a vaccine strain showed characteristic CPE at third passage level.

Induction of pluripotent stem cells from fibroblast cultures.

Science.gov (United States)

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Effect of silver nanoparticles and hydroxyproline, administered in ovo, on the development of blood vessels and cartilage collagen structure in chicken embryos

DEFF Research Database (Denmark)

Beck, Iwona; Hotowy, Anna; Sawosz, Ewa

2015-01-01

. An assessment of the mass of embryo and selected organs was carried out followed by measurements of the expression of the key signalling factors' fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A). Finally, an evaluation of collagen microstructure using scanning electron...... microscopy was performed. Our results clearly indicate that Hyp, Ag and AgHyp administered in ovo to chicken embryos did not harm embryos. Comparing to the control group, Hyp, Ag and the AgHyp complex significantly upregulated expression of the FGF-2 at the mRNA and protein levels. Moreover, Hyp, Ag and......It has been considered that concentrations of certain amino acids in the egg are not sufficient to fully support embryonic development of modern broilers. In this study we evaluated embryo growth and development with particular emphasis on one of the major components of connective tissue, collagen...

The effects of MRI on mouse embryos during fetal stage

Energy Technology Data Exchange (ETDEWEB)

Nakamura, Takashi; Sakazaki, Takahiko; Itokawa, Yuka [Suzuka University of Medical Science, Koriyama (Japan)] (and others)

2006-06-15

The effects of Magnetic Resonance Imaging (MRI) on mouse embryos at the early stage of organogenesis were investigated. Pregnant ICR mice were exposed on day 8 of gestation to MRI at 0.5 T for 0.5 hour to 3 hours. The mortality rates of embryos or fetuses, the incidence of external malformations, fetal body weight and sex ratio were observed at day 18 of gestation. A significant increase in embryonic mortality was observed after exposure to either 0.5 T MRI for 0.5 hour or 2 hours. However, the exposure to MRI for 1 hour or 3 hours did not induce any significant increase in embryonic mortality when compared with control. External malformations such as exencephaly, cleft palate and anomalies of tail were observed in all experimental groups exposed to each MRI. A statistically significant increase of external malformations was observed in all groups treated with 0.5 T MRI for 0.5 hour and 3 hours. The incidence of external malformations in the mice group exposed to 0.5 T MRI for 0.5-hour was found to be higher than those of mice group exposed to 0.5 T MRI for 2 hours. The effects of MRI on the external malformations might not to be dose-dependent. There was no statistically significant difference in fetal body weight and sex ratio among each MRI exposure groups.

Estradiol stimulation of inositolphospholipid metabolism in human endometrial fibroblasts

International Nuclear Information System (INIS)

Iida, K.; Imai, A.; Tamaya, T.

1989-01-01

Stimulated inositolphospholipid turnover has been proposed to constitute a signal-transducing mechanism in many cell types. To determine the inositolphospholipid turnover during stimulation by 17 beta-estradiol, the turnover kinetics of phospholipids was investigated in human endometrial fibroblasts. In cells incubated with [ 32 P] phosphate for 1 h, estradiol rapidly and persisitently (for at least 30 min) enhanced the rate of 32 P-labeling of phosphatidic acid (PA). On the other hand, after a lag time of 5 min, 32 P-labeling of phosphatidylinositol (PI) was also increased also. These sequential 32 P-labeling of PA and PI demonstrated that inositolphospholipid turnover was stimulated in fibroblasts exposed to estradiol. The rapid estrogen-stimulated inositolphospholipid turnover may not be through the mechanism associated with classical action of estrogen

The effects of pollutants on osmotic and ionic regulation of herring (Clupea harengus L. ) embryos and larvae

Energy Technology Data Exchange (ETDEWEB)

Ramsay, N.C. (Aberdeen Univ. (United Kingdom))

1991-01-01

This thesis looks at how osmoregulatory processes function during early ontogeny of herring (Clupea harengus L.) and how these can be affected by pollutants. First, during embryo osmoregulatory ontogeny, two distinct stages occur. Until the completion of epiboly and closure of the yolk plug, the embryo must rely wholly on passive osmoregulation. From epiboly to hatching, the embryo is able to regulate its osmolality and increases its water content through a drinking mechanism similar to that of the adult fish. Increased water content is paralleled by increased levels of solutes. The protein level thereafter decreases with subsequent increased ninhydrin positive substances. Excess salts are excreted through cells thought to be classical chloride cells. During the [open quotes]active[close quotes] osmoregulatory stage, the embryo has developed true osmotic homeostasis, providing continuity until the development of gills, skin, gut, kidney of the adult. Second, exposing embryos and larvae to a number of different pollutants affected parameters important for osmotic and ionic homeostasis. The most consistent response is an increased whole body electrolyte content and decreased water content, resulting in elevated osmolality. Exposure to drilling muds containing high levels of petrogenic components causes water loss from the larvae associated with a decreased drinking rate. Subsequent exposure to individual oil constituents at sublethal levels cause changes resulting in higher whole body electrolyte concentrations. In larvae exposed in vivo to metal levels below the Environmental Quality Standard, changes occurred in epithelial permeability. Effects on electrolyte concentrations also occurred in embryos but the mechanism was unclear. The mixture of heavy metals exposed in vitro resulted in a lower concentration required to cause a 50% reduction in whole body homogenate Na[sup +]K[sup +]-ATPase activity than for any individual metal.

Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

Science.gov (United States)

Nakayama, Asuka; Sato, Masahiro; Shinohara, Mariko; Matsubara, Shyuichiro; Yokomine, Takaaki; Akasaka, Eri; Yoshida, Mitsutoshi; Takao, Sonshin

2007-01-01

Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.

Towards more ecological relevance in sediment toxicity testing with fish: Evaluation of multiple bioassays with embryos of the benthic weatherfish (Misgurnus fossilis).

Science.gov (United States)

Schreiber, Benjamin; Fischer, Jonas; Schiwy, Sabrina; Hollert, Henner; Schulz, Ralf

2018-04-01

The effects of sediment contamination on fish are of high significance for the protection of ecosystems, human health and economy. However, standardized sediment bioassays with benthic fish species, that mimic bioavailability of potentially toxic compounds and comply with the requirements of alternative test methods, are still scarce. In order to address this issue, embryos of the benthic European weatherfish (Misgurnus fossilis) were exposed to freeze-dried sediment (via sediment contact assays (SCA)) and sediment extracts (via acute fish embryo toxicity tests) varying in contamination level. The extracts were gained by accelerated solvent extraction with (i) acetone and (ii) pressurized hot water (PHWE) and subsequently analyzed for polycyclic aromatic hydrocarbons, polychlorinated biphenyls and polychlorinated dibenzodioxins and dibenzofurans. Furthermore, embryos of the predominately used zebrafish (Danio rerio) were exposed to extracts from the two most contaminated sediments. Results indicated sufficient robustness of weatherfish embryos towards varying test conditions and sensitivity towards relevant sediment-bound compounds. Furthermore, a compliance of effect concentrations derived from weatherfish embryos exposed to sediment extracts (96h-LC 50 ) with both measured gradient of sediment contamination and previously published results was observed. In comparison to zebrafish, weatherfish embryos showed higher sensitivity to the bioavailability-mimicking extracts from PHWE but lower sensitivity to extracts gained with acetone. SCAs conducted with weatherfish embryos revealed practical difficulties that prevented an implementation with three of four sediments tested. In summary, an application of weatherfish embryos, using bioassays with sediment extracts from PHWE might increase the ecological relevance of sediment toxicity testing: it allows investigations using benthic and temperate fish species considering both bioavailable contaminants and animal welfare

Biological Screening of Newly Synthesized BIAN N-Heterocyclic Gold Carbene Complexes in Zebrafish Embryos

Science.gov (United States)

Farooq, Muhammad; Abu Taha, Nael; Butorac, Rachel R.; Evans, Daniel Anthony; Elzatahry, Ahmed A.; Elsayed, Elsayed Ahmed; Wadaan, Mohammad A. M.; Al-Deyab, Salem S.; Cowley, Alan H.

2015-01-01

N-Heterocyclic carbene (NHC) metal complexes possess diverse biological activities but have yet to be extensively explored as potential chemotherapeutic agents. We have previously reported the synthesis of a new class of NHC metal complexes N-heterocyclic with acetate [IPr(BIAN)AuOAc] and chloride [IPr(BIAN)AuCl] ligands. In the experiments reported herein, the zebrafish embryos were exposed to serial dilutions of each of these complexes for 10–12 h. One hundred percent mortality was observed at concentrations ≥50 µM. At sub-lethal concentrations (10–30 µM), both compounds influenced zebrafish embryonic development. However, quite diverse categories of abnormalities were found in exposed embryos with each compound. Severe brain deformation and notochord degeneration were evident in the case of [IPr(BIAN)AuOAc]. The zebrafish embryos treated with [IPr(BIAN)AuCl] exhibited stunted growth and consequently had smaller body sizes. A depletion of 30%–40% glutathione was detected in the treated embryos, which could account for one of the possible mechanism of neurotoxicity. The fact that these compounds are capable of both affecting the growth and also compromising antioxidant systems by elevating intracellular ROS production implies that they could play an important role as a new breed of therapeutic molecules. PMID:26501273

Hand-made cloned buffalo (Bubalus bubalis) embryos: comparison of different media and culture systems.

Science.gov (United States)

Shah, Riaz A; George, Aman; Singh, Manoj K; Kumar, Dharmendra; Chauhan, Manmohan S; Manik, Radhaysham; Palta, Prabhat; Singla, Suresh K

2008-12-01

Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.

Minute changes to the culture environment of mouse pre-implantation embryos affect the health of the conceptus

Directory of Open Access Journals (Sweden)

George Koustas

2016-07-01

Conclusions: Exposing mouse pre-implantation embryos to ambient air at 37.0 °C, even for brief periods for routine micromanipulations is detrimental to normal embryonic development. Our results highlight the importance of how small alterations in the culture environment can have major consequences for the health of the embryo.

Perturbation of the Developmental Potential of Preimplantation Mouse Embryos by Hydroxyurea

Directory of Open Access Journals (Sweden)

Edward R. Hills

2010-04-01

Experiments 2 and 3, (N = 10/Experiment folliculogenesis and ovulation were induced in untreated mice followed by mating. Recovered embryos were either exposed continuously (Experiment 2 or intermittently (Experiment 3 to bioavailable HU (18 μg HU/mL of WM + CZBt or WM + CZBt only (control. Treated mice sustained decreased ovarian wt, ovulation rate and circulating E2 compared with controls (P < 0.05. Fewer embryos retrieved from HU-treated mice developed to blastocyst stage (32% compared with those from controls (60%; P < 0.05. Furthermore, continuous or intermittent in vitro exposures of embryos to HU also resulted in reduced development to blastocyst stage (continuous HU, 9 vs. control, 63%; P < 0.05; intermittent HU, 20 vs. control, 62%; P < 0.05 with embryos exposed continuously to HU in vitro fairing worse. Even though HU is well tolerated, our data suggest that it compromises folliculogenesis and the ability of generated embryos to develop. Therefore, designed studies with larger numbers of patients receiving HU during pregnancy, with longer follow-up of exposed children and more careful assessment of embryo/fetotoxic effects, are required before this agent can be promoted as safe in pregnancy.

The Chromatin Regulator Brpf1 Regulates Embryo Development and Cell Proliferation*

Science.gov (United States)

You, Linya; Yan, Kezhi; Zou, Jinfeng; Zhao, Hong; Bertos, Nicholas R.; Park, Morag; Wang, Edwin; Yang, Xiang-Jiao

2015-01-01

With hundreds of chromatin regulators identified in mammals, an emerging issue is how they modulate biological and pathological processes. BRPF1 (bromodomain- and PHD finger-containing protein 1) is a unique chromatin regulator possessing two PHD fingers, one bromodomain and a PWWP domain for recognizing multiple histone modifications. In addition, it binds to the acetyltransferases MOZ, MORF, and HBO1 (also known as KAT6A, KAT6B, and KAT7, respectively) to promote complex formation, restrict substrate specificity, and enhance enzymatic activity. We have recently showed that ablation of the mouse Brpf1 gene causes embryonic lethality at E9.5. Here we present systematic analyses of the mutant animals and demonstrate that the ablation leads to vascular defects in the placenta, yolk sac, and embryo proper, as well as abnormal neural tube closure. At the cellular level, Brpf1 loss inhibits proliferation of embryonic fibroblasts and hematopoietic progenitors. Molecularly, the loss reduces transcription of a ribosomal protein L10 (Rpl10)-like gene and the cell cycle inhibitor p27, and increases expression of the cell-cycle inhibitor p16 and a novel protein homologous to Scp3, a synaptonemal complex protein critical for chromosome association and embryo survival. These results uncover a crucial role of Brpf1 in controlling mouse embryo development and regulating cellular and gene expression programs. PMID:25773539

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

Energy Technology Data Exchange (ETDEWEB)

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Pang, Daxin, E-mail: pdx@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China); Ouyang, Hongsheng, E-mail: ouyh@jlu.edu.cn [Jilin Province Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, 5333 Xi An DaLu, Changchun 130062 (China)

2011-07-29

Highlights: {yields} Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. {yields} The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. {yields} A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 {mu}g/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

Vitamin C enhances in vitro and in vivo development of porcine somatic cell nuclear transfer embryos

International Nuclear Information System (INIS)

Huang, Yongye; Tang, Xiaochun; Xie, Wanhua; Zhou, Yan; Li, Dong; Zhou, Yang; Zhu, Jianguo; Yuan, Ting; Lai, Liangxue; Pang, Daxin; Ouyang, Hongsheng

2011-01-01

Highlights: → Report for the first time that vitamin C has a beneficial effect on the development of porcine SCNT embryos. → The level of acH4K5 and Oct4 expression at blastocyst-stage was up-regulated after treatment. → A higher rate of gestation and increased number of piglets born were harvested in the treated group. -- Abstract: The reprogramming of differentiated cells into a totipotent embryonic state through somatic cell nuclear transfer (SCNT) is still an inefficient process. Previous studies revealed that the generation of induced pluripotent stem (iPS) cells from mouse and human fibroblasts could be significantly enhanced with vitamin C treatment. Here, we investigated the effects of vitamin C, to our knowledge for the first time, on the in vitro and in vivo development of porcine SCNT embryos. The rate of blastocyst development in SCNT embryos treated with 50 μg/mL vitamin C 15 h after activation (36.0%) was significantly higher than that of untreated SCNT embryos (11.5%). The enhanced in vitro development rate of vitamin C-treated embryos was associated with an increased acetylation level of histone H4 lysine 5 and higher Oct4, Sox2 and Klf4 expression levels in blastocysts, as determined by real-time PCR. In addition, treatment with vitamin C resulted in an increased pregnancy rate in pigs. These findings suggest that treatment with vitamin C is beneficial for enhancement of the in vitro and in vivo development of porcine SCNT embryos.

Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified.

Science.gov (United States)

Hammond, Elizabeth R; McGillivray, Brent C; Wicker, Sophie M; Peek, John C; Shelling, Andrew N; Stone, Peter; Chamley, Larry W; Cree, Lynsey M

2017-01-01

To characterize nuclear and mitochondrial DNA (mtDNA) in spent culture media from normally developing blastocysts to determine whether it could be used for noninvasive genetic assessment. Prospective embryo cohort study. Academic center and private in vitro fertilization (IVF) clinic. Seventy patients undergoing intracytoplasmic sperm injection (ICSI) and 227 blastocysts. Culture media assessment, artificial blastocoele fluid collapse and DNA analysis using digital polymerase chain reaction (dPCR), long-range PCR, quantitative PCR (qPCR), and DNA fingerprinting. Presence of nuclear and mtDNA in three different commercial culture media from Vitrolife and Irvine Scientific, spent embryo media assessment at the cleavage and blastocyst stages of development, and analysis of the internal media controls for each patient that had been exposed to identical conditions as embryo media but did not come into contact with embryos. Higher levels of nuclear and mtDNA were observed in the culture media that had been exposed to embryos compared with the internal media controls. Nuclear DNA (∼4 copies) and mtDNA (∼600 copies) could be detected in spent media, and the levels increased at the blastocyst stage. No increase in DNA was detected after artificial blastocoele fluid collapse. Mixed sex chromosome DNA was detected. This originated from contamination in the culture media and from maternal (cumulus) cells. Due to the limited amount of template, the presence of embryonic nuclear DNA could not be confirmed by DNA fingerprinting analysis. Currently DNA from culture media cannot be used for genetic assessment because embryo-associated structures release DNA into the culture medium and the DNA is of mixed origin. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos

International Nuclear Information System (INIS)

Li Dan; Lu Cailing; Wang Ju; Hu Wei; Cao Zongfu; Sun Daguang; Xia Hongfei; Ma Xu

2009-01-01

Arsenic usually accumulates in soil, water and airborne particles, from which it is taken up by various organisms. Exposure to arsenic through food and drinking water is a major public health problem affecting some countries. At present there are limited laboratory data on the effects of arsenic exposure on early embryonic development and the mechanisms behind its toxicity. In this study, we used zebrafish as a model system to investigate the effects of arsenite on early development. Zebrafish embryos were exposed to a range of sodium arsenite concentrations (0-10.0 mM) between 4 and 120 h post-fertilization (hpf). Survival and early development of the embryos were not obviously influenced by arsenite concentrations below 0.5 mM. However, embryos exposed to higher concentrations (0.5-10.0 mM) displayed reduced survival and abnormal development including delayed hatching, retarded growth and changed morphology. Alterations in neural development included weak tactile responses to light (2.0-5.0 mM, 30 hpf), malformation of the spinal cord and disordered motor axon projections (2.0 mM, 48 hpf). Abnormal cardiac function was observed as bradycardia (0.5-2.0 mM, 60 hpf) and altered ventricular shape (2.0 mM, 48 hpf). Furthermore, altered cell proliferation (2.0 mM, 24 hpf) and apoptosis status (2.0 mM, 24 and 48 hpf), as well as abnormal genomic DNA methylation patterning (2.0 mM, 24 and 48 hpf) were detected in the arsenite-treated embryos. All of these indicate a possible relationship between arsenic exposure and developmental failure in early embryogenesis. Our studies suggest that the negative effects of arsenic on vertebrate embryogenesis are substantial

Toxicity implications for early life stage Japanese medaka (Oryzias latipes) exposed to oxyfluorfen.

Science.gov (United States)

Powe, Doris K; Dasmahapatra, Asok K; Russell, Joseph L; Tchounwou, Paul B

2018-05-01

We investigated the potential toxic effects of Oxyfluorfen (OXY), an herbicide used in agriculture, on the embryo-larval development of Japanese medaka fish (Oryzias latipes). Embryos (1-day postfertilization) and larvae (2-day posthatch) were exposed to OXY (0.5-8 mg/L) for 96 h and evaluated for mortality and hatching on embryos, and the mortality and growth on larvae during depuration. It was observed that the embryo-mortality was inconsistently altered by OXY; only the 2 mg/L group showed significant reduction on embryo survivability. However, larval-mortality was concentration-dependent and OXY exposure induced scoliosis-like phenotypic features in the surviving larvae; the calculated LC 50 was 5.238 mg/L. Our data further indicated that larval skeleton, both axial and appendicular, was the potential target site of OXY. Skeletal growth in larvae exposed to 2 mg/L was inhibited significantly until 1 week of depuration with regard to the linear lengths of neurocranium, Meckel's cartilage, caudal vertebrae (first 10) in the axial skeletons, and pectoral fin and urostyle in the appendicular skeletons. Moreover, the total protein content remained unaltered by OXY after 96 h exposure; while the RNA concentration was reduced significantly in larvae exposed to 2 mg/L. Expression analysis of several genes by quantitative real-time RT-PCR (RT-qPCR) showed significant upregulation of zic5, a zinc-finger type transcription regulator, at the transcription level. This study indicated that the scoliosis induced by OXY in Japanese medaka larvae was the result of stunted skeletal growth, probably because of deregulation of zinc-finger type transcription regulators, at the genomic level. © 2018 Wiley Periodicals, Inc.

The influence of low-level radiation on nucleolus activity of cyprinidae embryos

International Nuclear Information System (INIS)

Arkhipchuk, V.V.

1990-01-01

The number and sizes of nucleoli in embryos of Cyprinus carpio and Carassius auratus gibelio exposed to low-level radiation at a dose-rate of 10 μ Gy/h and down were larger than those on non-irradiated controls

The use of the mouse chimera assay to detect early embryonic damage from male mice exposed to low-dose radiation

International Nuclear Information System (INIS)

Oudiz, D.; Warner, P.; Walsh, K.J.; Wiley, L.

1990-01-01

Mouse chimeras are in vitro aggregations of two 4-cell embryos and are used to detect subtle, nonlethal changes, which are expressed as a proliferative disadvantage in exposed embryos. One of the embryos is labeled with a viable dye (FITC) in order to determine the relative cellular contribution of each embryo when the chimera is dissociated 40 hours later. This proliferative disadvantage has been seen at doses which do not produce an effect on cell number when the embryos are cultured singly. Previously, the assay has detected a decrease in cellular proliferation in embryos from male mice exposed to a single dose of x-radiation as low as 0.05 Gy. In the current study, male mice were irradiated with a single dose of 0, 0.001, 0.01, or 0.05 Gy, and then serially mated for the next 8 weeks to unexposed females. Chimeras were constructed from control and treated embryos. Embryos from males treated with 0.05 Gy exhibited a significant decrease in cellular proliferation during weeks 6 and 7 post-irradiation. A similar decrease was seen in the males treated with 0.01 Gy. No reductions were observed from embryos cultured singly in any of the treatment groups

Low-dose prenatal alcohol exposure modulates weight gain and eliminates fractalkine expression in e14.5 mouse embryos

Directory of Open Access Journals (Sweden)

Jordyn Karliner

2017-07-01

Full Text Available Fetal Alcohol Spectrum Disorder (FASD is caused by maternal alcohol consumption during pregnancy and often leads to long-lasting developmental symptoms, including increased microglial migration and increased release of the chemokine, fractalkine, both of which play a role in embryonic brain development. However, the effects of low-dose alcohol exposure on microglia and fractalkine embryonically are not well documented. This study addresses this gap by using the voluntary drinking paradigm, Drinking in the Dark (DiD, to expose mice to acute doses of alcohol from embryonic day 7.5 (E7.5 to E14.5. Maternal mice and embryo analyses revealed increased embryo weights and a trend of increased gestational weight gain in alcohol-exposed mice compared to water-exposed mice. After quantifying soluble fractalkine concentrations through Western Blots, results indicated decreased fractalkine in alcohol-exposed mice compared to water-exposed. Overall, our data suggest that exposure to low doses of alcohol inhibits fractalkine release, which may affect microglial function.

DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

Energy Technology Data Exchange (ETDEWEB)

Zhang, Bingzhen [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Shen, Chunzi [Centers for Disease Control and Prevention, Zibo (China); Yang, Liu; Li, Chunhui; Yi, Anji [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Wang, Zhiping, E-mail: zhipingw@sdu.edu.cn [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China)

2013-12-01

Carbon disulfide (CS{sub 2}) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS{sub 2} via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD{sub 50} (157.85 mg/kg), 0.2 LD{sub 50} (315.7 mg/kg) and 0.4 LD{sub 50} (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS{sub 2}. - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss.

Cell surface response of chemically transformed, malignant mouse embryonal fibroblasts and human colon cancer cells to the maturation-promoting agent, N,N-dimethylformamide

International Nuclear Information System (INIS)

Marks, M.E.

1985-01-01

The lactoperoxidase/ 125 I radioiodination procedure was used to probe the cell surface of normal, nontransformed AKR-2B mouse embryo fibroblasts and malignant, permanently methylcholanthrene-transformed AKR-2B (AKR-MCA) cells to establish the relationship between cell surface changes and transformation/differentiation in this call system. AKR-MCA cells displayed surface alterations secondary to N,N-dimethylformamide (DFM)-promoted differentiation. Growth of AKR-MCA cells in DMF virtually eliminated the 85,000 and 63,000 molecular weight surface proteins susceptible to radioiodination and increased surface material of ∼200,000 molecular weight. Thus, surface profiles of DFM-treated AKR-MCA cells were essentially identical to those of nontransformed AKR-2B cells. Experimentation was extended to a cultured human colon cancer cell line (HCT MOSER). HCT MOSER cells exposed to DMF manifested marked, reversible morphological and surface changes which occurred as a function of time of growth in DMF and DMF concentration. Interestingly, material reactive with anti-fibronectin was found on the surfaces and in the culture medium of DFM-treated HCT MOSER cells

Repair of ultraviolet light damage to the DNA of cultured human epidermal keratinocytes and fibroblasts

International Nuclear Information System (INIS)

Taichman, L.B.; Setlow, R.B.

1979-01-01

Pure cultures of dermal fibroblasts and epidermal keroatinocytes have been obtained from a single biopsy of newborn foreskin. The cells were labeled, exposed to several doses of uv light, and allowed to repair in the dark for 16 h. The number of pyrimidine dimers before and after repair was assessed by measuring the numbers of sites in the DNA sensitive to a specific uv endonuclease. At all doses used, the extent of repair was similar in the cultured keratinocytes and cultured fibroblasts

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

Science.gov (United States)

Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.E.; Di Giulio, R.T.

2001-01-01

Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ???19.7??9.5 pg TCDD/??l (water bath) and 0.25??0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction. Copyright ?? 2001 Elsevier Science B.V.

Absence of nucleolus formation in raccoon dog-porcine interspecies somatic cell nuclear transfer embryos results in embryonic developmental failure.

Science.gov (United States)

Jeon, Yubyeol; Nam, Yeong-Hee; Cheong, Seung-A; Kwak, Seong-Sung; Lee, Eunsong; Hyun, Sang-Hwan

2016-08-25

Interspecies somatic cell nuclear transfer (iSCNT) can be a solution for preservation of endangered species that have limited oocytes. It has been reported that blastocyst production by iSCNT is successful even if the genetic distances between donors and recipients are large. In particular, domestic pig oocytes can support the development of canine to porcine iSCNT embryos. Therefore, we examined whether porcine oocytes may be suitable recipient oocytes for Korean raccoon dog iSCNT. We investigated the effects of trichostatin A (TSA) treatment on iSCNT embryo developmental patterns and nucleolus formation. Enucleated porcine oocytes were fused with raccoon dog fibroblasts by electrofusion and cleavage, and blastocyst development and nucleolus formation were evaluated. To our knowledge, this study is the first in which raccoon dog iSCNT was performed using porcine oocytes; we found that 68.5% of 158 iSCNT embryos had the ability to cleave. However, these iSCNT embryos did not develop past the 4-cell stage. Treatment with TSA did not affect iSCNT embryonic development; moreover, the nuclei failed to form nucleoli at 48 and 72 h post-activation (hpa). In contrast, pig SCNT embryos of the control group showed 18.8% and 87.9% nucleolus formation at 48 and 72 hpa, respectively. Our results demonstrated that porcine cytoplasts efficiently supported the development of raccoon dog iSCNT embryos to the 4-cell stage, the stage of porcine embryonic genome activation (EGA); however, these embryos failed to reach the blastocyst stage and showed defects in nucleolus formation.

Novel embryo selection techniques to increase embryo implantation in IVF attempts.

Science.gov (United States)

Sigalos, George Α; Triantafyllidou, Olga; Vlahos, Nikos F

2016-11-01

The final success of an IVF attempt depends on several steps and decisions taken during the ovarian stimulation, the oocyte retrieval, the embryo culture and the embryo transfer. The final selection of the embryos most likely to implant is the final step in this process and the responsibility of the lab. Apart from strict morphologic criteria that historically have been used in embryo selection, additional information on genetic, metabolomic and morphokinetic characteristics of the embryo is recently combined to morphology to select the embryo most likely to produce a pregnancy. In this manuscript, we review the most recent information on the current methods used for embryo selection presenting the predictive capability of each one. A literature search was performed on Pubmed, Medline and Cochrane Database of Systematic Reviews for published studies using appropriate key words and phrases with no limits placed on time. It seems that the combination of morphologic criteria in conjunction to embryo kinetics as documented by time-lapse technology provides the most reliable information on embryo quality. Blastocyst biopsy with subsequent comprehensive chromosome analysis allows the selection of the euploid embryos with the higher implantation potential. Embryo time-lapse imaging and blastocyst biopsy combined to comprehensive chromosome analysis are the most promising technologies to increase pregnancy rates and reduce the possibility of multiple pregnancies. However, further studies will demonstrate the capability of routinely using these technologies to significantly improve IVF outcomes.

Biological response of zebrafish embryos after short-term exposure to thifluzamide

Science.gov (United States)

Yang, Yang; Liu, Wenxian; Mu, Xiyan; Qi, Suzhen; Fu, Bin; Wang, Chengju

2016-12-01

Thifluzamide is a new amide fungicide, and its extensive application may have toxic effects on zebrafish. To better understand the underlying mechanism, we investigated in detail the potential toxic effects of thifluzamide on zebrafish embryos. In the present study, embryos were exposed to 0, 0.19, 1.90, and 2.85 mg/L thifluzamide for 4 days. Obvious pathological changes were found upon a histological exam, and negative changes in mitochondrial structure were observed under Transmission Electron Microscopy (TEM), which qualitatively noted the toxic effects of thifluzamide on embryos. Moreover, we quantitatively evaluated the enzyme activities [succinate dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), caspases], the contents of malonaldehyde (MDA) and interleukin-8 (IL-8) and the expression levels of the related genes. This study suggests that the negative changes in mitochondrial structure and SDH activity might be responsible for oxidative damage, cell apoptosis and inflammation, which would facilitate the action of these factors in cell death and might play a crucial role during toxic events. In addition to providing the first description of the mechanism of the toxic effects of thifluzamide on embryos, this study also represents a step towards using embryos to assess mitochondrial metabolism and disease.

Perflurooctanoic acid induces developmental cardiotoxicity in chicken embryos and hatchlings

International Nuclear Information System (INIS)

Jiang, Qixiao; Lust, Robert M.; Strynar, Mark J.; Dagnino, Sonia; DeWitt, Jamie C.

2012-01-01

Highlights: ► PFOA exposure thinned right ventricular wall thickness in D19 chicken embryo hearts. ► PFOA exposure induced left ventricle hypertrophy in hearts of hatchling chickens. ► PFOA exposure induced altered cardiac function in hatchling chickens. -- Abstract: Perfluorooctanoic acid (PFOA) is a widespread environmental contaminant that is detectable in serum of the general U.S. population. PFOA is a known developmental toxicant that induces mortality in mammalian embryos and is thought to induce toxicity via interaction with the peroxisome proliferator activated receptor alpha (PPARα). As the cardiovascular system is crucial for embryonic survival, PFOA-induced effects on the heart may partially explain embryonic mortality. To assess impacts of PFOA exposure on the developing heart in an avian model, we used histopathology and immunohistochemical staining for myosin to assess morphological alterations in 19-day-old chicken embryo hearts after PFOA exposure. Additionally, echocardiography and cardiac myofibril ATPase activity assays were used to assess functional alterations in 1-day-old hatchling chickens following developmental PFOA exposure. Overall thinning and thinning of a dense layer of myosin in the right ventricular wall were observed in PFOA-exposed chicken embryo hearts. Alteration of multiple cardiac structural and functional parameters, including left ventricular wall thickness, left ventricular volume, heart rate, stroke volume, and ejection fraction were detected with echocardiography in the exposed hatchling chickens. Assessment of ATPase activity indicated that the ratio of cardiac myofibril calcium-independent ATPase activity to calcium-dependent ATPase activity was not affected, which suggests that developmental PFOA exposure may not affect cardiac energetics. In summary, structural and functional characteristics of the heart appear to be developmental targets of PFOA, possibly at the level of cardiomyocytes. Additional studies will

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

International Nuclear Information System (INIS)

Han, Zhihua; Wang, Qiangwei; Fu, Jie; Chen, Hongshan; Zhao, Ye; Zhou, Bingsheng; Gong, Zhiyuan; Wei, Si; Li, Jun; Liu, Hongling; Zhang, Xiaowei; Liu, Chunsheng; Yu, Hongxia

2014-01-01

Highlights: • TBEP exposure decreased the survival of zebrafish embryos/larvae. • TBEP exposure led to its bioconcentration in zebrafish lavare. • TBEP caused developmental toxicity by inhibiting the degradation and utilization of nutrients. • TBEP exposure caused developmental toxicity by inducing apoptosis. - Abstract: The flame retardant tris(2-butoxyethyl) phosphate (TBEP) is a frequently detected contaminant in the environment, wildlife and human milk. The potentially toxic effects of TBEP and their underlying molecular mechanisms have not been elucidated. Here, zebrafish embryos were exposed to different concentrations of TBEP from 4 hours of post-fertilization (hpf) to 120 hpf, and effects on embryonic development and global protein expression patterns examined. Our results demonstrate that treatment with TBEP (0.8–100 mg/L) causes a concentration- and time-dependent decrease in embryonic survival and the hatching percentage. The median lethal concentration was 10.7 mg/L at 120 hpf. Furthermore, exposure to 150 or 800 μg/L TBEP inhibited the degradation and utilization of vitellogenins and down-regulated the expression of proteins related to cation binding, and lipid transport, uptake and metabolism, accompanied by a decrease in heart rate and body length. Exposure to TBEP (150 or 800 μg/L) also decreased the expression of proteins involved in cell proliferation and DNA repair, and led to an increased number of apoptotic cells in the tail region. Collectively, our results suggest that exposure to TBEP causes toxicity in the developing zebrafish by inhibiting the degradation and utilization of nutrients from the mother and inducing apoptosis

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate

Energy Technology Data Exchange (ETDEWEB)

Han, Zhihua [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Wang, Qiangwei [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Fu, Jie [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Chen, Hongshan [State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of the Environment, Northeast Normal University, Changchun 130024 (China); Department of Biological Sciences, National University of Singapore (Singapore); Zhao, Ye [Department of Biological Sciences, National University of Singapore (Singapore); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Gong, Zhiyuan [Department of Biological Sciences, National University of Singapore (Singapore); Wei, Si; Li, Jun; Liu, Hongling; Zhang, Xiaowei [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Liu, Chunsheng, E-mail: liuchunshengidid@126.com [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Department of Biological Sciences, National University of Singapore (Singapore); College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Yu, Hongxia, E-mail: yuhx@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China)

2014-05-01

Highlights: • TBEP exposure decreased the survival of zebrafish embryos/larvae. • TBEP exposure led to its bioconcentration in zebrafish lavare. • TBEP caused developmental toxicity by inhibiting the degradation and utilization of nutrients. • TBEP exposure caused developmental toxicity by inducing apoptosis. - Abstract: The flame retardant tris(2-butoxyethyl) phosphate (TBEP) is a frequently detected contaminant in the environment, wildlife and human milk. The potentially toxic effects of TBEP and their underlying molecular mechanisms have not been elucidated. Here, zebrafish embryos were exposed to different concentrations of TBEP from 4 hours of post-fertilization (hpf) to 120 hpf, and effects on embryonic development and global protein expression patterns examined. Our results demonstrate that treatment with TBEP (0.8–100 mg/L) causes a concentration- and time-dependent decrease in embryonic survival and the hatching percentage. The median lethal concentration was 10.7 mg/L at 120 hpf. Furthermore, exposure to 150 or 800 μg/L TBEP inhibited the degradation and utilization of vitellogenins and down-regulated the expression of proteins related to cation binding, and lipid transport, uptake and metabolism, accompanied by a decrease in heart rate and body length. Exposure to TBEP (150 or 800 μg/L) also decreased the expression of proteins involved in cell proliferation and DNA repair, and led to an increased number of apoptotic cells in the tail region. Collectively, our results suggest that exposure to TBEP causes toxicity in the developing zebrafish by inhibiting the degradation and utilization of nutrients from the mother and inducing apoptosis.

A reduced transcriptome approach to assess environmental toxicants using zebrafish embryo tests

Science.gov (United States)

This paper reports on the pilot testing of a new bioassay platform that monitors expression of 1600 genes in zebrafish embryos exposed to either single chemicals or complex water samples. The method provides a more cost effective, high throughput means to broadly evaluate the pot...

Maternal Diabetes Leads to Unphysiological High Lipid Accumulation in Rabbit Preimplantation Embryos

NARCIS (Netherlands)

Schindler, Maria; Pendzialek, Mareike; Santos, Alexander Navarrete; Ploesch, Torsten; Seyring, Stefanie; Guerke, Jacqueline; Haucke, Elisa; Knelangen, Julia Miriam; Fischer, Bernd; Santos, Anne Navarrete

According to the "developmental origin of health and disease" hypothesis, the metabolic set points of glucose and lipid metabolism are determined prenatally. In the case of a diabetic pregnancy, the embryo is exposed to higher glucose and lipid concentrations as early as during preimplantation

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

International Nuclear Information System (INIS)

Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

2014-01-01

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

Energy Technology Data Exchange (ETDEWEB)

Xu, Ting; Zhao, Jing [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Hu, Ping [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029 (China); Dong, Zhangji; Li, Jingyun [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); Zhang, Hongchang [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Yin, Daqiang, E-mail: yindq@tongji.edu.cn [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Zhao, Qingshun, E-mail: qingshun@nju.edu.cn [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China)

2014-06-01

Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

Energy Technology Data Exchange (ETDEWEB)

Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

2017-06-15

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

International Nuclear Information System (INIS)

Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

2017-01-01

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

Protein degradation in preimplantation mouse embryos and the lethality of tritiated amino acids

International Nuclear Information System (INIS)

Wielbold, J.L.

1982-01-01

The role of protein degradation in preimplantation development in the mouse was studied. Proteins of morulae and blastocysts (M and B) cultured in vitro after labeling for 1 hour (h) in 3 H-leucine exhibit a mean half-life (t 1 / 2 ) of 8.1 h. The t 1 / 2 tends to increase (9.5 h) when 10% fetal calf serum is added to the chase medium. This decrease in protein degradation in the presence of serum is associated with an increase in the percentage of B that are hatching (P 3 H-leucine in their proteins than did Day 4 embryos remaining in culture (P<0.02), while Day 4 embryos in a Day 3 uterus retained the same amount of radioactivity as did Day 4 embryos in culture. This differential effect of uterine environment was also seen when Day 4 embryos were transferred to recipients. More fetuses developed to term when the recipient was in Day 3 of PSP (50.8%) than when the recipient was in Day 4 PSP (25.9%, P<0.001), regardless of the age of the recipient. Age of the recipient does affect the percentage of transferred embryos developing to term. Thus, protein degradation may vary with the stage of embryo development and the conditions to which the embryos are exposed. However, even low levels of incorporated tritiated leucine can have lethal effects on the embryos and compromise the validity of the protein half-lives determined

Proteome Changes in Maize Embryo (Zea mays L) Induced by Ion Beam Implantation Treatment

International Nuclear Information System (INIS)

Li Yongliang; Qin Guangyong; Huo Yuping; Tian Shuangqi; Tang Jihua

2009-01-01

Low energy ion beam implantation was applied to the maize (Zea mays L) embryo proteome using two-dimensional gel electrophoresis. Protein profile analysis detected more than 1100 protein spots, 72 of which were determined to be expressed differently in the treated and control (not exposed to ion beam implantation) embryos. Of the 72 protein spots, 53 were up-regulated in the control and 19 were more abundantly expressed in the ion beam-treated embryos. The spots of up- or down-regulated proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Among the identified proteins, 11 were up-regulated in the treated embryos. Four of these up-regulated proteins were antioxidant molecules, three were related to stress response, two to sugar metabolism and two were associated with heat shock response. Of the five proteins up-regulated in the control embryos, three were functionally related to carbohydrate metabolism; the functions of the remaining two proteins were unknown. The data collected during this study indicate that treatment of maize embryos with low energy ion beam implantation induces changes in stress tolerance enzymes/proteins, possibly as a result of alterations in metabolism. (ion beam bioengineering)

Proteome Changes in Maize Embryo (Zea mays L) Induced by Ion Beam Implantation Treatment

Science.gov (United States)

Li, Yongliang; Tang, Jihua; Qin, Guangyong; Huo, Yuping; Tian, Shuangqi

2009-08-01

Low energy ion beam implantation was applied to the maize (Zea mays L) embryo proteome using two-dimensional gel electrophoresis. Protein profile analysis detected more than 1100 protein spots, 72 of which were determined to be expressed differently in the treated and control (not exposed to ion beam implantation) embryos. Of the 72 protein spots, 53 were up-regulated in the control and 19 were more abundantly expressed in the ion beam-treated embryos. The spots of up- or down-regulated proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Among the identified proteins, 11 were up-regulated in the treated embryos. Four of these up-regulated proteins were antioxidant molecules, three were related to stress response, two to sugar metabolism and two were associated with heat shock response. Of the five proteins up-regulated in the control embryos, three were functionally related to carbohydrate metabolism; the functions of the remaining two proteins were unknown. The data collected during this study indicate that treatment of maize embryos with low energy ion beam implantation induces changes in stress tolerance enzymes/proteins, possibly as a result of alterations in metabolism.

How the embryo makes a limb: determination, polarity and identity.

Science.gov (United States)

Tickle, Cheryll

2015-10-01

The vertebrate limb with its complex anatomy develops from a small bud of undifferentiated mesoderm cells encased in ectoderm. The bud has its own intrinsic polarity and can develop autonomously into a limb without reference to the rest of the embryo. In this review, recent advances are integrated with classical embryology, carried out mainly in chick embryos, to present an overview of how the embryo makes a limb bud. We will focus on how mesoderm cells in precise locations in the embryo become determined to form a limb and express the key transcription factors Tbx4 (leg/hindlimb) or Tbx5 (wing/forelimb). These Tbx transcription factors have equivalent functions in the control of bud formation by initiating a signalling cascade involving Wnts and fibroblast growth factors (FGFs) and by regulating recruitment of mesenchymal cells from the coelomic epithelium into the bud. The mesoderm that will form limb buds and the polarity of the buds is determined with respect to both antero-posterior and dorso-ventral axes of the body. The position in which a bud develops along the antero-posterior axis of the body will also determine its identity - wing/forelimb or leg/hindlimb. Hox gene activity, under the influence of retinoic acid signalling, is directly linked with the initiation of Tbx5 gene expression in the region along the antero-posterior axis of the body that will form wings/forelimbs and determines antero-posterior polarity of the buds. In contrast, Tbx4 expression in the regions that will form legs/hindlimbs is regulated by the homeoprotein Pitx1 and there is no evidence that Hox genes determine antero-posterior polarity of the buds. Bone morphogenetic protein (BMP) signalling determines the region along the dorso-ventral axis of the body in which both wings/forelimbs and legs/hindlimbs develop and dorso-ventral polarity of the buds. The polarity of the buds leads to the establishment of signalling regions - the dorsal and ventral ectoderm, producing Wnts and BMPs

Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

Directory of Open Access Journals (Sweden)

Ryutaro Hirasawa

Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

Patterns of some extracellular matrix gene expression are similar in cells from cleft lip-palate patients and in human palatal fibroblasts exposed to diazepam in culture

International Nuclear Information System (INIS)

Marinucci, Lorella; Balloni, Stefania; Bodo, Maria; Carinci, Francesco; Pezzetti, Furio; Stabellini, Giordano; Carmela, Conte; Lumare, Eleonora

2009-01-01

Prenatal exposure to diazepam, a prototype sedative drug that belongs to Benzodiazepines, can lead to orofacial clefting in human newborns. By using real-time PCR, in the present study we investigated whether diazepam elicits gene expression alterations in extracellular matrix (ECM) components, growth factors and gamma-aminobutyric acid receptor (GABRB3), implicated in the coordinate regulation of palate development. Palate fibroblasts were treated with diazepam (Dz-N fibroblasts) and compared to cleft lip-palate (CLP) fibroblasts obtained from patients with no known exposure to diazepam or other teratogens. Untreated fibroblasts from non-CLP patients were used as control. The results showed significant convergences in gene expression pattern of collagens, fibromodulin, vitronectin, tenascin C, integrins and metalloprotease MMP13 between Dz-N and CLP fibroblasts. Among the growth factors, constitutive Fibroblast Growth Factor 2 (FGF2) was greatly enhanced in Dz-N and CLP fibroblasts and associated with a higher reduction of FGF receptor. Transforming Growth Factor beta 3 (TGFβ 3 ) resulted up-regulated in CLP fibroblasts and decreased in Dz-N fibroblasts. We found phenotypic differences exhibited by Dz-N and CLP fibroblasts in GABRB3 gene regulation, so further studies are necessary to determine whether GABAergic system could be involved in the development of diazepam mediated CLP phenotype. Taken together the results elucidate the molecular mechanisms underlying possible toxicology effects induced by diazepam. Counselling of women on the safety of diazepam exposure is clinically important, also for the forensic consequences

Effects of tributyltin (TBT) on Xenopus tropicalis embryos at environmentally relevant concentrations.

Science.gov (United States)

Guo, Suzhen; Qian, Lijuan; Shi, Huahong; Barry, Terence; Cao, Qinzhen; Liu, Junqi

2010-04-01

Tributyltin (TBT) has been widely used as a biocide in antifouling paints and is a known endocrine disrupting chemical. In this paper, we exposed embryos of Xenopus tropicalis to 50-400ngL(-1) tributyltin chloride. TBT significantly decreased the survival rate, reduced the body length and retarded the development of embryos after 24, 36 and 48h of exposure. These effects of TBT were concentration- and time-dependent. Embryos treated with TBT showed multiple malformations. The most obvious alterations were abnormal eyes, enlarged proctodaeum, narrow fins, and skin hypopigmentation. Enlarged proctodaeum and narrow fins were mainly observed after 36 and 48h of exposure. The loss of eye pigmentation or the absence of external eyes occurred after 24 and 36h of exposure, while extended lenses or edemas of eyes were more commonly observed after 48h of exposure. Additional malformations included: small anterior region of heads, pericardial edemas, enlarged trunks, and bent tails. These results suggested that TBT is very toxic to X. tropicalis embryos at environmentally relevant concentrations.

[Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

Science.gov (United States)

Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

2008-10-14

To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

Polycyclic aromatic hydrocarbons and dibutyl phthalate disrupt dorsal-ventral axis determination via the Wnt/{beta}-catenin signaling pathway in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Fairbairn, Elise A., E-mail: efairbairn@ucdavis.edu [University of California Davis, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923 (United States); Bonthius, Jessica, E-mail: jessica.bonthius@gmail.com [University of California Davis, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923 (United States); Cherr, Gary N., E-mail: gncherr@ucdavis.edu [University of California Davis, Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923 (United States); Department of Environmental Toxicology, University of California Davis, Davis, CA 95616 (United States); Department of Nutrition, University of California Davis, Davis, CA 95616 (United States)

2012-11-15

The canonical Wnt/{beta}-catenin signaling pathway is critical during early teleost development for establishing the dorsal-ventral axis. Within this pathway, GSK-3{beta}, a key regulatory kinase in the Wnt pathway, regulates {beta}-catenin degradation and thus the ability of {beta}-catenin to enter nuclei, where it can activate expression of genes that have been linked to the specification of the dorsal-ventral axis. In this study, we describe the morphological abnormalities that resulted in zebrafish embryos when axis determination was disrupted by environmental contaminants. These abnormalities were linked to abnormal nuclear accumulation of {beta}-catenin. Furthermore, we demonstrated that the developmental abnormalities and altered nuclear {beta}-catenin accumulation occurred when embryos were exposed to commercial GSK-3{beta} inhibitors. Zebrafish embryos were exposed to commercially available GSK-3 inhibitors (GSK-3 Inhibitor IX and 1-azakenpaullone), or common environmental contaminants (dibutyl phthalate or the polycyclic aromatic hydrocarbons phenanthrene and fluorene) from the 2 to 8-cell stage through the mid-blastula transition (MBT). These embryos displayed morphological abnormalities at 12.5 h post-fertilization (hpf) that were comparable to embryos exposed to lithium chloride (LiCl) (300 mM LiCl for 10 min, prior to the MBT), a classic disruptor of embryonic axis determination. Whole-mount immunolabeling and laser scanning confocal microscopy were used to localize {beta}-catenin. The commercial GSK-3 Inhibitors as well as LiCl, dibutyl phthalate, fluorene and phenanthrene all induced an increase in the levels of nuclear {beta}-catenin throughout the embryo, indicating that the morphological abnormalities were a result of disruption of Wnt/{beta}-catenin signaling during dorsal-ventral axis specification. The ability of environmental chemicals to directly or indirectly target GSK-3{beta} was assessed. Using Western blot analysis, the ability of these

Determining the pharmacological activity of Physalis peruviana fruit juice on rabbit eyes and fibroblast primary cultures.

Science.gov (United States)

Pardo, Juan Manuel; Fontanilla, Marta Raquel; Ospina, Luis Fernando; Espinosa, Lady

2008-07-01

The pharmacologic activity of compounds isolated from Physalis peruviana has been demonstrated. The use of this fruit juice for treating pterygium has been reported in Colombian traditional medicine. However, studies demonstrating the fruit juice's pharmacologic activity when used in this disease have not been published to date. In the present study the anti-inflammatory and cytostatic activities of P. peruviana fruit juice in a rabbit eye inflammatory model were investigated. A novel rabbit eye inflammation model was developed for studying the juice's anti-inflammatory activity (based on an adaptation of the Draize test). Cytostatic activity was evaluated by measuring and comparing growth rates of cultured fibroblasts exposed and not exposed to various fruit juice concentrations. P. peruviana fruit juice exhibited a mild anti-inflammatory activity compared with methylprednisolone, a known anti-inflammatory drug. An interesting dose-dependent cytostatic effect on cultured fibroblasts was also established. The data found suggest that the P. peruviana fruit juice anti-pterygium effect described in traditional medicine may be related to its inhibiting fibroblast growth. The present study contributes to the pharmacologic knowledge regarding a remedy commonly used in Colombian traditional medicine.

Fibroblastic rheumatism

Directory of Open Access Journals (Sweden)

Jyoti Ranjan Parida

2017-01-01

Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

Effect of titanium dioxide nanoparticles on zebrafish embryos and developing retina

Directory of Open Access Journals (Sweden)

Ya-Jie Wang

2014-12-01

Full Text Available AIM:To investigate the impact of titanium dioxide nanoparticles (TiO2 NPs on embryonic development and retinal neurogenesis. METHODS:The agglomeration and sedimentation of TiO2 NPs solutions at different dilutions were observed, and the ultraviolet-visible spectra of their supernatants were measured. Zebrafish embryos were experimentally exposed to TiO2 NPs until 72h postfertilization (hpf. The retinal neurogenesis and distribution of the microglia were analyzed by immunohistochemistry and whole mount in situ hybridization. RESULTS: The1 mg/L was determined to be an appropriate exposure dose. Embryos exposed to TiO2 NPs had a normal phenotype. The neurogenesis was initiated on time, and ganglion cells, cones and rods were well differentiated at 72 hpf. The expression of fms mRNA and the 4C4 antibody, which were specific to microglia in the central nervous system (CNS, closely resembled their endogenous profile. CONCLUSION:These data demonstrate that short-term exposure to TiO2 NPs at a low dose does not lead to delayed embryonic development or retinal neurotoxicity.

Effects of rare earth elements La and Yb on the morphological and functional development of zebrafish embryos

Institute of Scientific and Technical Information of China (English)

Jun'an Cui; Zhiyong Zhang; Wei Bai; Ligang Zhang; Xiao He; Yuhui Ma; Yan Liu; Zhifang Chai

2012-01-01

In recent years,with the wide applications and mineral exploitation of rare earth elements,their potential environmental and health effects have caused increasing public concern.Effect of rare earth elements La and Yb on the morphological and functional development of zebrafish embryos were studied.The embryos were exposed to La3+ or Yb3+ at 0,0.01,0.1,0.3,0.5 and 1.0 mmol/L,respectively.Early life stage parameters such as egg and embryo mortality,gastrula development,tail detachment,eyes,somite formation,circulatory system,pigmentation,malformations,hatching rate,length of larvae and mortality were investigated.The results showed La3+ and Yb3+ delayed zebrafish embryo and larval development,decreased survival and hatching rates,and caused tail malformation in a concentration-dependent way.Moreover,heavy rare-earth ytterbium led to more severe acute toxicity of zebrafish embryo than light rare-earth lanthanum.

Absorbed dose conversion coefficients for embryo and foetus in neutron fields

International Nuclear Information System (INIS)

Chen, J.

2007-01-01

The Monte Carlo code MCNPX has been used to determine mean absorbed doses to the embryo and foetus when the mother is exposed to neutron fields. There are situations, such as on-board aircraft, where high-energy neutrons are often peaked in top down (TOP) direction. In addition to previous publications for standard irradiation geometries, this study provides absorbed dose conversion coefficients for the embryo of 8 weeks and the foetus of 3, 6 or 9 months at TOP irradiation geometry. The conversion coefficients are compared with the coefficients in isotropic irradiation (ISO). With increasing neutron energies, the conversion coefficients in TOP irradiation become dominant. A set of conversion coefficients is constructed from the higher value in either ISO or TOP irradiation at a given neutron energy. In cases where the irradiation geometry is not adequately known, this set of conversion coefficients can be used in a conservative dose assessment for embryo and foetus in neutron fields. (authors)

Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin

DEFF Research Database (Denmark)

Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mollerup, Sarah

2017-01-01

of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.......BACKGROUND: Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety...... of neoplasms but their role in BCC is poorly understood. METHODS: Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical...

Cancer associated fibroblasts (CAFs) are activated in cutaneous basal cell carcinoma and in the peritumoural skin

DEFF Research Database (Denmark)

Omland, Silje Haukali; Wettergren, Erika Elgstrand; Mourier, Tobias

2017-01-01

of chemokines involved in tumour progression and immunosuppression (CXCL12, CCL17). Fibroblasts from chronically sun-exposed skin near tumours show gene expression patterns resembling that of CAFs, indicating that stromal fibroblasts in cancer-free surgical BCC margins exhibit a tumour promoting phenotype.......Background: Cutaneous basal cell carcinoma (BCC) is the commonest cancer worldwide. BCC is locally invasive and the surrounding stromal microenvironment is pivotal for tumourigenesis. Cancer associated fibroblasts (CAFs) in the microenvironment are essential for tumour growth in a variety...... of neoplasms but their role in BCC is poorly understood. Methods: Material included facial BCC and control skin from the peritumoural area and from the buttocks. With next-generation sequencing (NGS) we compared mRNA expression between BCC and peritumoural skin. qRT-PCR, immunohistochemical...

N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

LENUS (Irish Health Repository)

Burke, John P

2012-02-01

BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

Lessons from Embryos: Haeckel's Embryo Drawings, Evolution, and Secondary Biology Textbooks

Science.gov (United States)

Wellner, Karen L.

2014-01-01

In 1997, developmental biologist Michael Richardson compared his research team's embryo photographs to Ernst Haeckel's 1874 embryo drawings and called Haeckel's work "noncredible". "Science" soon published "Haeckel's Embryos: Fraud Rediscovered," and Richardson's comments further reinvigorated criticism of Haeckel by…

[Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

Science.gov (United States)

Zhao, H; Teng, X M; Li, Y F

2017-11-25

Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

Noninvasive embryo assessment technique based on buoyancy and its association with embryo survival after cryopreservation.

Science.gov (United States)

Wessels, Cara; Penrose, Lindsay; Ahmad, Khaliq; Prien, Samuel

2017-11-01

Embryo cryopreservation offers many benefits by allowing genetic preservation, genetic screening, cost reduction, global embryo transport and single embryo transfer. However, freezing of embryos decreases embryo viability, as intracellular ice crystal formation often damages embryos. Success rates of frozen embryo transfer are expected to be 15-20% less than fresh embryo transfer. We have developed a noninvasive embryo assessment technique (NEAT) which enables us to predict embryo viability based on buoyancy. The purpose of this research was twofold. First was to determine if a NEAT, through a specific gravity device can detect embryo survival of cryopreservation. Second, it was to relate embryo buoyancy to embryo viability for establishing pregnancies in sheep. Blastocysts descent times were measured on one-hundred sixty-nine mice blastocysts before cryopreservation, according to standard protocol and post-thawing blastocysts descent times were measured again. There was a significant difference in blastocyst post-thaw descent times with NEAT in those blastocysts which demonstrated viability from those that did not (P embryos. Further studies on a larger scale commercial setting will evaluate the efficacy of NEAT. Copyright © 2017 Elsevier Inc. All rights reserved.

Evaluation of MWNT toxic effects on daphnia and zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Olasagasti, Maider; Rainieri, Sandra [AZTI-TECNALIA, Parque Tecnologico de Bizkaia 609, 48160 Derio (Spain)], E-mail: srainieri@azti.es; Alvarez, Noelia; Vera, Carolina [INASMET-TECNALIA, Mikeletegi pasealekua, 2, Parque Tecnologico, 20009 San Sebastian (Spain)

2009-05-01

Organisms of daphnia (Daphnia magna) and zebrafish (Danio rerio) embryos were exposed to a range of different concentrations of COOH-functionalized MWCNT suspended in an aqueous solution of Tween 20. Immobilization of daphnia and growth retardation, inhibition and malformation of zebrafish embryos were the endpoints tested after 24 and 48 hours. Immobilization of daphnia could be observed from 3 to 16 ppm and an increasing mortality of zebrafish embryo was detected at all the concentration tested. To identify more subtle toxic effects, we took advantage of the extensive information available on the zebrafish genome and monitored by RT-PCR the expression patterns of different zebrafish genes that could act as toxicity bio-markers. At some of the concentrations tested, changes in the expression profiles of the genes examined were detected. Our results suggest that MWCNT could potentially represent a risk to human health and environment, therefore a wider range of concentrations and further testing of this molecules should be carried out to define possible limitations in their use.

Entire mesodermal mantle behaves as Spemann's organizer in dorsoanterior enhanced Xenopus laevis embryos

International Nuclear Information System (INIS)

Kao, K.R.; Elinson, R.P.

1988-01-01

The body plan of Xenopus laevis can be respecified by briefly exposing early cleavage stage embryos to lithium. Such embryos develop exaggerated dorsoanterior structures such as a radial eye and cement gland. In this paper, we demonstrate that the enhanced dorsoanterior phenotype results from an overcommitment of mesoderm to dorsoanterior mesoderm. Histological and immunohistochemical observations reveal that the embryos have a greatly enlarged notochord with very little muscle tissue. In addition, they develop a radial, beating heart, suggesting that lithium also specifies anterior mesoderm and pharyngeal endoderm. Randomly oriented diametrically opposed marginal zone grafts from lithium-treated embryos, when transplanted into ultraviolet (uv)-irradiated axis-deficient hosts, rescue dorsal axial structures. These transplantation experiments demonstrate that the entire marginal zone of the early gastrula consists of presumptive dorsal mesoderm. Vital dye marking experiments also indicate that the entire marginal zone maps to the prominent proboscis that is composed of chordamesoderm and represents the long axis of the embryo. These results suggest that lithium respecifies the mesoderm of Xenopus laevis embryos so that it differentiates into the Spemann organizer. We suggest that the origin of the dorsoanterior enhanced phenotypes generated by lithium and the dorsoanterior deficient phenotypes generated by uv irradiation are due to relative quantities of organizer. Our evidence demonstrates the existence of a continuum of body plan phenotypes based on this premise

Miniaturized embryo array for automated trapping, immobilization and microperfusion of zebrafish embryos.

Directory of Open Access Journals (Sweden)

Jin Akagi

Full Text Available Zebrafish (Danio rerio has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP. The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Embryo density may affect embryo quality during in vitro culture in a microwell group culture dish.

Science.gov (United States)

Lehner, Adam; Kaszas, Zita; Murber, Akos; Rigo, Janos; Urbancsek, Janos; Fancsovits, Peter

2017-08-01

Culturing embryos in groups is a common practice in mammalian embryology. Since the introduction of different microwell dishes, it is possible to identify oocytes or embryos individually. As embryo density (embryo-to-volume ratio) may affect the development and viability of the embryos, the purpose of this study was to assess the effect of different embryo densities on embryo quality. Data of 1337 embryos from 228 in vitro fertilization treatment cycles were retrospectively analyzed. Embryos were cultured in a 25 μl microdrop in a microwell group culture dish containing 9 microwells. Three density groups were defined: Group 1 with 2-4 (6.3-12.5 μl/embryo), Group 2 with 5-6 (4.2-5.0 μl/embryo), and Group 3 with 7-9 (2.8-3.6 μl/embryo) embryos. Proportion of good quality embryos was higher in Group 2 on both days (D2: 18.9 vs. 31.5 vs. 24.7%; p Culturing 5-6 embryos together in a culture volume of 25 μl may benefit embryo quality. As low egg number, position, and distance of the embryos may influence embryo quality, results should be interpreted with caution.

Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization

Energy Technology Data Exchange (ETDEWEB)

Barranger, Audrey, E-mail: audrey.barranger@ifremer.fr [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’Ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France); Benabdelmouna, Abdellah, E-mail: abdellah.benabdelmouna@ifremer.fr [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Dégremont, Lionel [Ifremer, SG2M, Laboratory of Genetics and Pathology of Marine Molluscs, Avenue de Mus du Loup, 17390 La Tremblade (France); Burgeot, Thierry; Akcha, Farida [Ifremer, Department of Biogeochemistry and Ecotoxicology, Laboratory of Ecotoxicology, Rue de l’Ile d’Yeu, BP 21105, 44311 Nantes Cedex 03 (France)

2015-02-15

Highlights: • FISH was realized on oyster embryos from diuron-exposed genitors. • rDNA genes were used as probes on the interphase nuclei of embryo preparations. • Higher aneuploidy level was observed in embryos from diuron-exposed genitors. • Hypo- and hyperdiploid (triploid) nuclei were detected. - Abstract: Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto Crassostrea gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment.

Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization

International Nuclear Information System (INIS)

Barranger, Audrey; Benabdelmouna, Abdellah; Dégremont, Lionel; Burgeot, Thierry; Akcha, Farida

2015-01-01

Highlights: • FISH was realized on oyster embryos from diuron-exposed genitors. • rDNA genes were used as probes on the interphase nuclei of embryo preparations. • Higher aneuploidy level was observed in embryos from diuron-exposed genitors. • Hypo- and hyperdiploid (triploid) nuclei were detected. - Abstract: Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto Crassostrea gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment

The polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

Science.gov (United States)

Zhang, Yujiang; Zhan, Songmei; Cao, Pengli; Liu, Ning; Chen, Xuehong; Wang, Yuejun; Wang, Chunbo

2005-09-01

To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25% 1%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant proerty.

Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

Science.gov (United States)

Akshey, Y S; Malakar, D; De, A Kumar; Jena, M Kumar; Pawar, S Kumar; Dutta, R; Sahu, S

2011-05-01

The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos. Copyright © 2011 Elsevier Inc. All rights reserved.

Potential protective effect of L-cysteine against the toxicity of acrylamide and furan in exposed Xenopus laevis embryos: an interaction study.

Science.gov (United States)

Williams, John Russell; Rayburn, James R; Cline, George R; Sauterer, Roger; Friedman, Mendel

2014-08-06

The embryo toxicities of two food-processing-induced toxic compounds, acrylamide and furan, with and without added L-cysteine were examined individually and in mixtures using the frog embryo teratogenesis assay-Xenopus (FETAX). The following measures of developmental toxicity were used: (a) 96 h LC50, the median concentration causing 50% embryo lethality; (b) 96 h EC50, the median concentration causing 50% malformations of the surviving embryos; and (c) teratogenic index (96 h LC50/96 h EC50), an estimate of teratogenic risk. Calculations of toxic units (TU) were used to assess possible antagonism, synergism, or response addition of several mixtures. The evaluated compounds demonstrated counterintuitive effects. Furan had lower than expected toxicity in Xenopus embryos and, unlike acrylamide, does not seem to be teratogenic. However, the short duration of the tests may not show the full effects of furan if it is truly primarily genotoxic and carcinogenic. L-Cysteine showed unexpected properties in the delay of hatching of the embryos. The results from the interaction studies between combination of two or three components (acrylamide plus L-cysteine; furan plus L-cysteine; acrylamide plus furan; acrylamide plus furan and L-cysteine) show that furan and acrylamide seem to have less than response addition at 1:1 toxic unit ratio in lethality. Acrylamide and L-cysteine show severe antagonism even at low 19 acrylamide/1 L-cysteine TU ratios. Data from the mixture of acrylamide, furan, and L-cysteine show a slight antagonism, less than would have been expected from binary mixture exposures. Bioalkylation mechanisms and their prevention are discussed. There is a need to study the toxicological properties of mixtures of acrylamide and furan concurrently formed in heat-processed food.

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

Science.gov (United States)

McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Toxicity of Buprofezin on the Survival of Embryo and Larvae of African Catfish, Clarias gariepinus (Bloch)

OpenAIRE

Marimuthu, Kasi; Muthu, Narmataa; Xavier, Rathinam; Arockiaraj, Jesu; Rahman, M. Aminur; Subramaniam, Sreeramanan

2013-01-01

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatme...

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract.

Science.gov (United States)

Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

2016-12-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. Copyright © 2016. Published by Elsevier B.V.

Photoreactivation rescue and dark repair demonstrated in UV-irradiated embryos of the self-fertilizing fish Rivulus ocellatus marmoratus (Teleostei; Aplocheilidae)

International Nuclear Information System (INIS)

Park, E.-H.; Yi, A.-K.

1989-01-01

The effects of photoreactivation (PR) rescue and dark repair on the survival of UV-irradiated embryos of the hermaphroditic fish (Rivulus ocellatus marmoratus) are reported. When UV-irradiated embryos were illuminated by photoreactivating light (PRL) from fluorescent lamps, survival at the hatching stage was markedly increased. The maximum recovery to UV damage was shown by embryos that were exposed to PRL for at least 6 h after UV irradiation. The effect of PRL decreased 30 min after UV irradiation and not PR rescue ws detected beyond 96 h. Treatment with 2 mM caffeine for 48 h after UV irradiation increased the sensitivity of the embryos in the dark. The above results demonstrate that Rivulus embryos have an efficient PR system and a caffeine-sensitive dark repair capacity. (author). 31 refs.; 5 figs

Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

International Nuclear Information System (INIS)

Basso, F G; Pansani, T N; Turrioni, A P S; Hebling, J; De Souza Costa, C A; Kurachi, C; Bagnato, V S

2013-01-01

Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm −2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal–Wallis and Mann–Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions. (paper)

Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

Science.gov (United States)

Basso, F. G.; Pansani, T. N.; Turrioni, A. P. S.; Kurachi, C.; Bagnato, V. S.; Hebling, J.; de Souza Costa, C. A.

2013-05-01

Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate—zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype—LaserTABLE (InGaAsP—780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions.

Quantitation of the repair of gamma-radiation-induced double-strand DNA breaks in human fibroblasts

International Nuclear Information System (INIS)

Woods, W.G.

1981-01-01

The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a tsub(1/2) of 10 min for repair of 5000 rad exposure and 39 min for repair of 10000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial fast and a later slow mechanism may be involved. (Auth.)

Muscular contractions in the zebrafish embryo are necessary to reveal thiuram-induced notochord distortions

International Nuclear Information System (INIS)

Teraoka, Hiroki; Urakawa, Satsuki; Nanba, Satomi; Nagai, Yuhki; Wu Dong; Imagawa, Tomohiro; Tanguay, Robert L.; Svoboda, Kurt; Handley-Goldstone, Heather M.; Stegeman, John J.; Hiraga, Takeo

2006-01-01

Dithiocarbamates form a large group of chemicals that have numerous uses in agriculture and medicine. It has been reported that dithiocarbamates, including thiuram (tetramethylthiuram disulfide), cause wavy distortions of the notochord in zebrafish and other fish embryos. In the present study, we investigated the mechanism underlying the toxicity of thiuram in zebrafish embryos. When embryos were exposed to thiuram (2-1000 nM: 0.48-240 μg/L) from 3 h post fertilization (hpf) (30% epiboly) until 24 hpf (Prim-5), all embryos develop wavy notochords, disorganized somites, and have shortened yolk sac extensions. The thiuram response was specific and did not cause growth retardation or mortality at 24 hpf. The thiuram-dependent responses showed the same concentration dependence with a waterborne EC 5 values of approximately 7 nM. Morphometric measurements revealed that thiuram does not affect the rate of notochord lengthening. However, the rate of overall body lengthening was significantly reduced in thiuram-exposed animals. Other dithiocarbamates, such as ziram, caused similar malformations to thiuram. While expression of genes involved in somitogenesis was not affected, the levels of notochord-specific transcripts were altered after the onset of malformations. Distortion of the notochord started precisely at 18 hpf, which is concomitant with onset of spontaneous rhythmic trunk contractions. Abolishment of spontaneous contractions using tricaine, α-bungarotoxin, and a paralytic mutant sofa potato, resulted in normal notochord morphology in the presence of thiuram. These results indicate that muscle activity is necessary to reveal the underlying functional deficit and suggest that the developmental target of dithiocarbamates impairs trunk plasticity through an unknown mechanism

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

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Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

2016-07-01

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

Science.gov (United States)

Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

2015-04-01

Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A novel embryo culture media supplement that improves pregnancy rates in mice.

Science.gov (United States)

Highet, A R; Bianco-Miotto, T; Pringle, K G; Peura, A; Bent, S; Zhang, J; Nottle, M B; Thompson, J G; Roberts, C T

2017-03-01

The preimplantation embryo in vivo is exposed to numerous growth factors in the female reproductive tract, which are not recapitulated in embryo culture media in vitro The IGF2 and plasminogen activator systems facilitate blastocyst development. We hypothesized that the addition of IGF2 in combination with urokinase plasminogen activator (uPA) and plasminogen could improve rates of blastocyst hatching and implantation in mice. B6BcF1 and CBAB6F2 mouse embryos were divided into one of four supplemented culture media treatment groups: (1) control (media only); (2) 12.5 nM IGF2; (3) 10 µg/mL uPA and 5 µg/mL plasminogen; or (4) a combination of IGF2, uPA and plasminogen treatments. Embryo development to blastocyst stage and hatching were assessed before transfer to pseudopregnant recipient females and implantation, pregnancy rates and postnatal growth were assessed. After 90.5 h of culture, IGF2 + U + P treatment increased the percentage of B6BcF1 embryos that were hatching/hatched and percentage developing to blastocyst stage compared with controls (P culture, IGF2, uPA and plasminogen supplementation of culture media can improve pregnancy success, but the effect of treatment is dependent on the mouse strain. © 2017 Society for Reproduction and Fertility.

Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

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Rong Yao

Full Text Available Pulmonary fibrosis is one of the most common complications of paraquat (PQ poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR. Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR 1 small-interfering RNA (siRNA group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8 and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05. Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05. APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a

[Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

Science.gov (United States)

Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

2014-06-01

To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

International Nuclear Information System (INIS)

Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

1982-01-01

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

Induction of cytochrome P450 1 genes and stress response genes in developing zebrafish exposed to ultraviolet radiation

Energy Technology Data Exchange (ETDEWEB)

Behrendt, Lars [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Joensson, Maria E. [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Department of Environmental Toxicology, Uppsala University (Sweden); Goldstone, Jared V. [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States)

2010-06-01

Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48 h post-fertilization (hpf). Embryos exposed for 2, 4 or 6 h twice over 2 days to UVB (0.62 W/m{sup 2}; 8.9-26.7 kJ/m{sup 2}) plus UVA (2.05 W/m{sup 2}; 29.5-144.6 kJ/m{sup 2}) had moderately (2.4 {+-} 0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1 {+-} 0.2 and 2.3 {+-} 0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m{sup 2} UVB (26.8 kJ/m{sup 2}). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m{sup 2}) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m{sup 2}). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8 dpf) to UVB at 0.93 W/m{sup 2} (26.8 kJ/m{sup 2}) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

Potential of human twin embryos generated by embryo splitting in assisted reproduction and research.

Science.gov (United States)

Noli, Laila; Ogilvie, Caroline; Khalaf, Yacoub; Ilic, Dusko

2017-03-01

Embryo splitting or twinning has been widely used in veterinary medicine over 20 years to generate monozygotic twins with desirable genetic characteristics. The first human embryo splitting, reported in 1993, triggered fierce ethical debate on human embryo cloning. Since Dolly the sheep was born in 1997, the international community has acknowledged the complexity of the moral arguments related to this research and has expressed concerns about the potential for reproductive cloning in humans. A number of countries have formulated bans either through laws, decrees or official statements. However, in general, these laws specifically define cloning as an embryo that is generated via nuclear transfer (NT) and do not mention embryo splitting. Only the UK includes under cloning both embryo splitting and NT in the same legislation. On the contrary, the Ethics Committee of the American Society for Reproductive Medicine does not have a major ethical objection to transferring two or more artificially created embryos with the same genome with the aim of producing a single pregnancy, stating that 'since embryo splitting has the potential to improve the efficacy of IVF treatments for infertility, research to investigate the technique is ethically acceptable'. Embryo splitting has been introduced successfully to the veterinary medicine several decades ago and today is a part of standard practice. We present here an overview of embryo splitting experiments in humans and non-human primates and discuss the potential of this technology in assisted reproduction and research. A comprehensive literature search was carried out using PUBMED and Google Scholar databases to identify studies on embryo splitting in humans and non-human primates. 'Embryo splitting' and 'embryo twinning' were used as the keywords, alone or in combination with other search phrases relevant to the topics of biology of preimplantation embryos. A very limited number of studies have been conducted in humans and non

Effects of environmental and artificial UV-B radiation on freshwater prawn Macrobrachium olfersi embryos

Energy Technology Data Exchange (ETDEWEB)

Nazari, Evelise Maria [Programa de Pos-Graduacao em Ciencias Morfologicas, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, 21949-902 Rio de Janeiro, RJ (Brazil); Universidade Federal de Santa Catarina, Departamento de Biologia Celular, Embriologia e Genetica, Campus Universitario, 88040-900 Florianopolis, SC (Brazil); Ammar, Dib [Universidade do Oeste de Santa Catarina, Departamento de Biologia, Campus Universitario, 89600-000 Joacaba, SC (Brazil); Bem, Andreza Fabro de; Latini, Alexandra [Universidade Federal de Santa Catarina, Departamento de Bioquimica, Campus Universitario, 88040-900 Florianopolis, SC (Brazil); Mueller, Yara Maria Rauh [Universidade Federal de Santa Catarina, Departamento de Biologia Celular, Embriologia e Genetica, Campus Universitario, 88040-900 Florianopolis, SC (Brazil); Allodi, Silvana, E-mail: sallodi@histo.ufrj.br [Programa de Pos-Graduacao em Ciencias Morfologicas, Instituto de Ciencias Biomedicas, Universidade Federal do Rio de Janeiro, 21949-902 Rio de Janeiro, RJ (Brazil)

2010-06-01

The recent decrease of the stratospheric ozone has resulted in an increase of ultraviolet-B (UV-B) radiation reaching the Earth's surface. In freshwater ecosystems with transparent water, UV-B rays easily penetrate and potentially cause harmful effects to organisms. In this study, embryos of the prawn Macrobrachium olfersi were used to evaluate the impact of UV-B rays in freshwater environments. We observed three groups of embryos: the first was to assess whether UV-B radiation produced morphological defects and/or biochemical impairments in the laboratory. The second was to check whether embryos with the same impairments as those observed in the laboratory were found in their environment, under natural solar radiation. The third group was the non-irradiated control. The embryos irradiated with 310 mW cm{sup -2} UV-B for 30 min showed morphological alterations similar to those observed in embryos from the environmental control group. The most important effects of the UV-B radiation observed in M. olfersi embryos were morphological (1.2% of the total number of embryos from the environment and 2.8% of the total number of irradiated embryos), pigmentation changes in the eyes (78.0% of the total number of embryos from the environment and 98.9% of the total number of irradiated embryos), and disruption of the chromatophores (46.9% of the total number of embryos from the environment and 95.5% of the total number of irradiated embryos). We also observed an increase in egg volume, which was accompanied by a significant increase in water content in UV-B irradiated groups when compared with aquaria control embryos. In addition, a significant decrease in the mitotic index in eggs exposed to UV-B radiation was detected (0.17 for the embryos from the aquaria control, 0.10 for the embryos of the environmental control, and 0.04 for the irradiated groups). The low levels of NPSH and high levels of TBARS indicated that UV-B rays directly compromised the antioxidant function of

Effects of environmental and artificial UV-B radiation on freshwater prawn Macrobrachium olfersi embryos

International Nuclear Information System (INIS)

Nazari, Evelise Maria; Ammar, Dib; Bem, Andreza Fabro de; Latini, Alexandra; Mueller, Yara Maria Rauh; Allodi, Silvana

2010-01-01

The recent decrease of the stratospheric ozone has resulted in an increase of ultraviolet-B (UV-B) radiation reaching the Earth's surface. In freshwater ecosystems with transparent water, UV-B rays easily penetrate and potentially cause harmful effects to organisms. In this study, embryos of the prawn Macrobrachium olfersi were used to evaluate the impact of UV-B rays in freshwater environments. We observed three groups of embryos: the first was to assess whether UV-B radiation produced morphological defects and/or biochemical impairments in the laboratory. The second was to check whether embryos with the same impairments as those observed in the laboratory were found in their environment, under natural solar radiation. The third group was the non-irradiated control. The embryos irradiated with 310 mW cm -2 UV-B for 30 min showed morphological alterations similar to those observed in embryos from the environmental control group. The most important effects of the UV-B radiation observed in M. olfersi embryos were morphological (1.2% of the total number of embryos from the environment and 2.8% of the total number of irradiated embryos), pigmentation changes in the eyes (78.0% of the total number of embryos from the environment and 98.9% of the total number of irradiated embryos), and disruption of the chromatophores (46.9% of the total number of embryos from the environment and 95.5% of the total number of irradiated embryos). We also observed an increase in egg volume, which was accompanied by a significant increase in water content in UV-B irradiated groups when compared with aquaria control embryos. In addition, a significant decrease in the mitotic index in eggs exposed to UV-B radiation was detected (0.17 for the embryos from the aquaria control, 0.10 for the embryos of the environmental control, and 0.04 for the irradiated groups). The low levels of NPSH and high levels of TBARS indicated that UV-B rays directly compromised the antioxidant function of the

Effects of photodynamic therapy on dermal fibroblasts from xeroderma pigmentosum and Gorlin-Goltz syndrome patients.

Science.gov (United States)

Zamarrón, Alicia; García, Marta; Río, Marcela Del; Larcher, Fernando; Juarranz, Ángeles

2017-09-29

PDT is widely applied for the treatment of non-melanoma skin cancer pre-malignant and malignant lesions (actinic keratosis, basal cell carcinoma and in situ squamous cell carcinoma). In photodynamic therapy (PDT) the interaction of a photosensitizer (PS), light and oxygen leads to the formation of reactive oxygen species (ROS) and thus the selective tumor cells eradication. Xeroderma pigmentosum (XP) and Gorlin-Goltz Syndrome (GS) patients are at high risk of developing skin cancer in sun-exposed areas. Therefore, the use of PDT as a preventive treatment may constitute a very promising therapeutic modality for these syndromes. Given the demonstrated role of cancer associated fibroblasts (CAFs) in tumor progression and the putative CAFs features of some cancer-prone genodermatoses fibroblasts, in this study, we have further characterized the phenotype of XP and GS dermal fibroblasts and evaluated their response to methyl-δ-aminolevulinic acid (MAL)-PDT compared to that of dermal fibroblasts obtained from healthy donors. We show here that XP/GS fibroblasts display clear features of CAFs and present a significantly higher response to PDT, even after being stimulated with UV light, underscoring the value of this therapeutic approach for these rare skin conditions and likely to other forms of skin cancer were CAFs play a major role.

Site-Directed Genome Knockout in Chicken Cell Line and Embryos Can Use CRISPR/Cas Gene Editing Technology

Directory of Open Access Journals (Sweden)

Qisheng Zuo

2016-06-01

Full Text Available The present study established an efficient genome editing approach for the construction of stable transgenic cell lines of the domestic chicken (Gallus gallus domesticus. Our objectives were to facilitate the breeding of high-yield, high-quality chicken strains, and to investigate gene function in chicken stem cells. Three guide RNA (gRNAs were designed to knockout the C2EIP gene, and knockout efficiency was evaluated in DF-1 chicken fibroblasts and chicken ESCs using the luciferase single-strand annealing (SSA recombination assay, T7 endonuclease I (T7EI assay, and TA clone sequencing. In addition, the polyethylenimine-encapsulated Cas9/gRNA plasmid was injected into fresh fertilized eggs. At 4.5 d later, frozen sections of the embryos were prepared, and knockout efficiency was evaluated by the T7EI assay. SSA assay results showed that luciferase activity of the vector expressing gRNA-3 was double that of the control. Results of the T7EI assay and TA clone sequencing indicated that Cas9/gRNA vector-mediated gene knockdown efficiency was approximately 27% in both DF-1 cells and ESCs. The CRISPR/Cas9 vector was also expressed in chicken embryos, resulting in gene knockdown in three of the 20 embryos (gene knockdown efficiency 15%. Taken together, our results indicate that the CRISPR/Cas9 system can mediate stable gene knockdown at the cell and embryo levels in domestic chickens.

Human cytomegalovirus replicates in gamma-irradiated fibroblasts

International Nuclear Information System (INIS)

Shanley, J.D.

1986-01-01

Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication

Embryo aggregation does not improve the development of interspecies somatic cell nuclear transfer embryos in the horse.

Science.gov (United States)

Gambini, Andrés; De Stéfano, Adrián; Jarazo, Javier; Buemo, Carla; Karlanian, Florencia; Salamone, Daniel Felipe

2016-09-01

The low efficiency of interspecies somatic cell nuclear transfer (iSCNT) makes it necessary to investigate new strategies to improve embryonic developmental competence. Embryo aggregation has been successfully applied to improve cloning efficiency in mammals, but it remains unclear whether it could also be beneficial for iSCNT. In this study, we first compared the effect of embryo aggregation over in vitro development and blastocyst quality of porcine, bovine, and feline zona-free (ZF) parthenogenetic (PA) embryos to test the effects of embryo aggregation on species that were later used as enucleated oocytes donors in our iSCNT study. We then assessed whether embryo aggregation could improve the in vitro development of ZF equine iSCNT embryos after reconstruction with porcine, bovine, and feline ooplasm. Bovine- and porcine-aggregated PA blastocysts had significantly larger diameters compared with nonaggregated embryos. On the other hand, feline- and bovine-aggregated PA embryos had higher blastocyst cell number. Embryo aggregation of equine-equine SCNT was found to be beneficial for embryo development as we have previously reported, but the aggregation of three ZF reconstructed embryos did not improve embryo developmental rates on iSCNT. In vitro embryo development of nonaggregated iSCNT was predominantly arrested around the stage when transcriptional activation of the embryonic genome is reported to start on the embryo of the donor species. Nevertheless, independent of embryo aggregation, equine blastocyst-like structures could be obtained in our study using domestic feline-enucleated oocytes. Taken together, these results reported that embryo aggregation enhance in vitro PA embryo development and embryo quality but effects vary depending on the species. Embryo aggregation also improves, as expected, the in vitro embryo development of equine-equine SCNT embryos; however, we did not observe positive effects on equine iSCNT embryo development. Among oocytes

Developmental toxicity of cartap on zebrafish embryos.

Science.gov (United States)

Zhou, Shengli; Dong, Qiaoxiang; Li, Shaonan; Guo, Jiangfeng; Wang, Xingxing; Zhu, Guonian

2009-12-13

Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144h post-fertilization, to a range of concentrations of 25-1000microg/l. Results of the experiment indicated that cartap concentrations of 100microg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25microg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

International Nuclear Information System (INIS)

Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen

2016-01-01

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

Energy Technology Data Exchange (ETDEWEB)

Ju, Yan; Liu, Chunying; Lu, Wenwen; Zhang, Quan; Sodmergen, E-mail: sodmergn@pku.edu.cn

2016-05-27

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysis revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.

Toxicity of cypermethrin on the embryo and larvae of Gangetic mystus, Mystus cavasius

NARCIS (Netherlands)

Ali, Md Haider; Sumon, Kizar Ahmed; Sultana, Marin; Rashid, Harunur

2018-01-01

The objective of the present study was to elucidate the effects of cypermethrin on the embryo and the larvae of Gangetic mystus, Mystus cavasius. Therefore, fertilized eggs (n = 100) and 1-day-old larvae (n = 100) were exposed to six different concentrations of cypermethrin (0, 2, 4, 8, 16 and 32 μg

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

Science.gov (United States)

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

International Nuclear Information System (INIS)

Bringas, J.E.; Caceres, J.L.

1996-01-01

Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

Effect of low-power red light laser irradiation on the viability of human skin fibroblast

Energy Technology Data Exchange (ETDEWEB)

Bednarska, K.; Rozga, B.; Leyko, W.; Bryszewska, M. [Institute of Biophysics, University of Lodz (Poland); Kolodziejczyk, K.; Szosland, D. [Diabetological Clinic, Medical Academy of Lodz (Poland)

1998-10-01

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm{sup 2}). The energy densities were 2 J/cm{sup 2} and 12 J/cm{sup 2}, respectively, and the irradiation was carried out at a temperature of 22 C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 C, in the presence of 5% CO{sub 2}, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation. (orig.) With 4 figs., 2 tabs., 13 refs.

Embryos, individuals, and persons: an argument against embryo creation and research.

Science.gov (United States)

Tollefsen, C

2001-01-01

One strategy for arguing that it should be legally permissible to create human embryos, or to use spare human embryos, for scientific research purposes involves the claim that such embryos cannot be persons because they are not human individuals while twinning may yet take place. Being a human individual is considered to be by most people a necessary condition for being a human person. I argue first that such an argument against the personhood of embryos must be rationally conclusive if their destruction in public places such as laboratories is to be countenanced. I base this argument on a popular understanding of the role that the notion of privacy plays in abortion laws. I then argue that such arguments against personhood are not rationally conclusive. The claim that the early embryos is not a human individual is not nearly as obvious as some assert.

EXPOSURE TO A P13KINASE INHIBITOR PRODUCED DYSMORPHOGENESIS IN NEURULATION-STAGED MOUSE EMBRYOS IN CULTURE

Science.gov (United States)

The haloacetic acids (HAA) are a family of chemicals that are drinking water disinfection byproducts. We previously reported that bromo- and chloro-acetic acids alter embryonic development when mouse conceptuses are directly exposed to these xenobiotics in whole embryo culture. C...

Single-embryo transfer versus multiple-embryo transfer.

Science.gov (United States)

Gerris, Jan

2009-01-01

Despite the progress made in assisted reproductive technology, live birth rates remain disappointingly low. Multiple-embryo transfer has been an accepted practice with which to increase the success rate. This has led to a higher incidence of multiple-order births compared with natural conception, which not only increase the risk of mortality and morbidity to both mother and children but are also associated with social and economic consequences. Elective single-embryo transfer (eSET) was developed in an effort to increase singleton pregnancies in assisted reproduction. Studies comparing eSET with multiple-embryo transfer highlight the benefit of this approach and suggest that, with careful patient selection and the transfer of good-quality embryos, the risk of a multiple-order pregnancy can be reduced without significantly decreasing live birth rates. Although the use of eSET has gradually increased in clinical practice, its acceptance has been limited by factors such as availability of funding and awareness of the procedure. An open discussion of eSET is warranted in an effort to enable a broader understanding by physicians and patients of the merits of this approach. Ultimately, eSET may provide a more cost-effective, potentially safer approach to patients undergoing assisted reproduction technology.

Mouse Embryo Compaction.

Science.gov (United States)

White, M D; Bissiere, S; Alvarez, Y D; Plachta, N

2016-01-01

Compaction is a critical first morphological event in the preimplantation development of the mammalian embryo. Characterized by the transformation of the embryo from a loose cluster of spherical cells into a tightly packed mass, compaction is a key step in the establishment of the first tissue-like structures of the embryo. Although early investigation of the mechanisms driving compaction implicated changes in cell-cell adhesion, recent work has identified essential roles for cortical tension and a compaction-specific class of filopodia. During the transition from 8 to 16 cells, as the embryo is compacting, it must also make fundamental decisions regarding cell position, polarity, and fate. Understanding how these and other processes are integrated with compaction requires further investigation. Emerging imaging-based techniques that enable quantitative analysis from the level of cell-cell interactions down to the level of individual regulatory molecules will provide a greater understanding of how compaction shapes the early mammalian embryo. © 2016 Elsevier Inc. All rights reserved.

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test.

Science.gov (United States)

Hallare, Arnold V; Ruiz, Paulo Lorenzo S; Cariño, J C Earl D

2014-05-01

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Polycyclic aromatic hydrocarbons affect survival and development of common snapping turtle (Chelydra serpentina) embryos and hatchlings

Energy Technology Data Exchange (ETDEWEB)

Van Meter, Robin J [School of Environmental Science, Engineering, and Policy and Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Spotila, James R [School of Environmental Science, Engineering, and Policy and Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States); Avery, Harold W [School of Environmental Science, Engineering, and Policy and Department of Bioscience and Biotechnology, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104 (United States)

2006-08-15

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds found in the John Heinz National Wildlife Refuge in Philadelphia, Pennsylvania. We assessed the impact of PAHs and crude oil on snapping turtle development and behavior by exposing snapping turtle eggs from the Refuge and from three clean reference sites to individual PAHs or a crude oil mixture at stage 9 of embryonic development. Exposure to PAHs had a significant effect on survival rates in embryos from one clean reference site, but not in embryos from the other sites. There was a positive linear relationship between level of exposure to PAHs and severity of deformities in embryos collected from two of the clean reference sites. Neither righting response nor upper temperature tolerance (critical thermal maximum, CTM) of snapping turtle hatchlings with no or minor deformities was significantly affected by exposure to PAHs. - Exposure to polycyclic aromatic hydrocarbons on the egg reduces survival of snapping turtle embryos and causes developmental abnormalities.

Polycyclic aromatic hydrocarbons affect survival and development of common snapping turtle (Chelydra serpentina) embryos and hatchlings

International Nuclear Information System (INIS)

Van Meter, Robin J.; Spotila, James R.; Avery, Harold W.

2006-01-01

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds found in the John Heinz National Wildlife Refuge in Philadelphia, Pennsylvania. We assessed the impact of PAHs and crude oil on snapping turtle development and behavior by exposing snapping turtle eggs from the Refuge and from three clean reference sites to individual PAHs or a crude oil mixture at stage 9 of embryonic development. Exposure to PAHs had a significant effect on survival rates in embryos from one clean reference site, but not in embryos from the other sites. There was a positive linear relationship between level of exposure to PAHs and severity of deformities in embryos collected from two of the clean reference sites. Neither righting response nor upper temperature tolerance (critical thermal maximum, CTM) of snapping turtle hatchlings with no or minor deformities was significantly affected by exposure to PAHs. - Exposure to polycyclic aromatic hydrocarbons on the egg reduces survival of snapping turtle embryos and causes developmental abnormalities

Interspecies somatic cell nucleus transfer with porcine oocytes as recipients: A novel bioassay system for assessing the competence of canine somatic cells to develop into embryos.

Science.gov (United States)

Sugimura, S; Narita, K; Yamashiro, H; Sugawara, A; Shoji, T; Terashita, Y; Nishimori, K; Konno, T; Yoshida, M; Sato, E

2009-09-01

Interspecies somatic cell nucleus transfer (iSCNT) could be a useful bioassay system for assessing the ability of mammalian somatic cells to develop into embryos. To examine this possibility, we performed canine iSCNT using porcine oocytes, allowed to mature in vitro, as recipients. Canine fibroblasts from the tail tips and dewclaws of a female poodle (Fp) and a male poodle (Mp) were used as donors. We demonstrated that the use of porcine oocytes induced blastocyst formation in the iSCNT embryos cultured in porcine zygote medium-3. In Fp and Mp, the rate of blastocyst formation from cleaved embryos (Fp: 6.3% vs. 22.4%; and Mp: 26.1% vs. 52.4%) and the number of cells at the blastocyst stage (Fp: 30.7 vs. 60.0; and Mp: 27.2 vs. 40.1) were higher in the embryos derived from dewclaw cells than in those derived from tail-tip cells (Ptip cells of Fp. Only blastocysts derived from dewclaw cells of Mp developed outgrowths. However, outgrowth formation was retrieved in the embryos derived from dewclaw cells of Fp by aggregation at the 4-cell stage. We inferred that iSCNT performed using porcine oocytes as recipients could represent a novel bioassay system for evaluating the developmental competence of canine somatic cells.

TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

Science.gov (United States)

Kucab, Jill E; Zwart, Edwin P; van Steeg, Harry; Luijten, Mirjam; Schmeiser, Heinz H; Phillips, David H; Arlt, Volker M

2016-03-01

3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of advanced glycation end-products on sun-exposed and non-exposed cutaneous sites during the ageing process in humans.

Science.gov (United States)

Crisan, Maria; Taulescu, Marian; Crisan, Diana; Cosgarea, Rodica; Parvu, Alina; Cãtoi, Cornel; Drugan, Tudor

2013-01-01

The glycation process is involved in both the intrinsic (individual, genetic) and extrinsic (ultraviolet light, polution and lifestyle) aging processes, and can be quantified at the epidermal or dermal level by histological, immunohistochemical (IHC), or imagistic methods. Our study is focused on a histological and immunohistological comparison of sun-protected regions versus sun-exposed regions from different age groups of skin phototype III subjects, related to the aging process. Skin samples collected from non-protected and UV protected regions of four experimental groups with different ages, were studied using histology and IHC methods for AGE-CML [N(epsilon)-(carboxymethyl)lysine]. A semi-quantitative assessment of the CML expression in the microvascular endothelium and dermal fibroblasts was performed. The Pearson one-way ANOVA was used to compare data between the groups. In the dermis of sun-exposed skin, the number and the intensity of CML positive cells in both fibroblasts and endothelial cells (p<0.05) was higher compared to sun-protected skin, and was significantly increased in older patients. The sun-exposed areas had a more than 10% higher AGE-CML score than the protected areas. No statistically significant correlation was observed between the histological score and the IHC expression of CML. We concluded that in healthy integument, the accumulation of final glycation products increases with age and is amplified by ultraviolet exposure. The study provides new knowledge on differences of AGE-CML between age groups and protected and unprotected areas and emphasizes that endothelium and perivascular area are most affected, justifying combined topical and systemic therapies.

Expression of advanced glycation end-products on sun-exposed and non-exposed cutaneous sites during the ageing process in humans.

Directory of Open Access Journals (Sweden)

Maria Crisan

Full Text Available The glycation process is involved in both the intrinsic (individual, genetic and extrinsic (ultraviolet light, polution and lifestyle aging processes, and can be quantified at the epidermal or dermal level by histological, immunohistochemical (IHC, or imagistic methods. Our study is focused on a histological and immunohistological comparison of sun-protected regions versus sun-exposed regions from different age groups of skin phototype III subjects, related to the aging process. Skin samples collected from non-protected and UV protected regions of four experimental groups with different ages, were studied using histology and IHC methods for AGE-CML [N(epsilon-(carboxymethyllysine]. A semi-quantitative assessment of the CML expression in the microvascular endothelium and dermal fibroblasts was performed. The Pearson one-way ANOVA was used to compare data between the groups. In the dermis of sun-exposed skin, the number and the intensity of CML positive cells in both fibroblasts and endothelial cells (p<0.05 was higher compared to sun-protected skin, and was significantly increased in older patients. The sun-exposed areas had a more than 10% higher AGE-CML score than the protected areas. No statistically significant correlation was observed between the histological score and the IHC expression of CML. We concluded that in healthy integument, the accumulation of final glycation products increases with age and is amplified by ultraviolet exposure. The study provides new knowledge on differences of AGE-CML between age groups and protected and unprotected areas and emphasizes that endothelium and perivascular area are most affected, justifying combined topical and systemic therapies.

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Science.gov (United States)

Sun, Yanyan; Zhang, Yanli; Wang, Ziyu; Song, Yang; Wang, Feng

2013-01-01

Background Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. PMID:24204972

Effects of temperature on gene expression in embryos of the coral Montastraea faveolata

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Randall Carly J

2009-12-01

Full Text Available Abstract Background Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5°C, 29.0°C, and 31.5°C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours. Results Analysis of differentially expressed genes indicated that increased temperatures may lead to oxidative stress, apoptosis, and a structural reconfiguration of the cytoskeletal network. Metabolic processes were downregulated, and the action of histones and zinc finger-containing proteins may have played a role in the long-term regulation upon heat stress. Conclusions Embryos responded differently depending on exposure time and temperature level. Embryos showed expression of stress-related genes already at a temperature of 29.0°C, but seemed to be able to counteract the initial response over time. By contrast, embryos at 31.5°C displayed continuous expression of stress genes. The genes that played a role in the response to elevated temperatures consisted of both highly conserved and coral-specific genes. These genes might serve as a basis for research into coral-specific adaptations to stress responses and global climate change.

Assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

International Nuclear Information System (INIS)

Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Miller, L.; Samuels, S.J.; Chang, R.J.; Overstreet, J.W.

1988-01-01

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos

The multiple stressor effect in zebrafish embryos from simultaneous exposure to ionising radiation and cadmium

International Nuclear Information System (INIS)

Ng, C Y P; Choi, V W Y; Lam, A C L; Yu, K N; Cheng, S H

2013-01-01

Living organisms are exposed to a mixture of environmental stressors, and the resultant effects are referred to as multiple stressor effects. In the present work, we studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to ionising radiation (alpha particles) and cadmium through quantification of apoptotic signals at 24 h postfertilisation (hpf) revealed by vital dye acridine orange staining. For each set of experiments, 32–40 dechorionated embryos were deployed, which were divided into four groups each having 8–10 embryos. The four groups of embryos were referred to as (1) the control group (C), which received no further treatments after dechorionation; (2) the Cd-dosed and irradiated group (CdIr), which was exposed to 100 μM Cd from 5 to 24 hpf, and also received about 4.4 mGy from alpha particles at 5 hpf; (3) the irradiated group (Ir), which received about 4.4 mGy from alpha particles at 5 hpf; and (4) the Cd-dosed group (Cd), which was exposed to 100 μM Cd from 5 to 24 hpf. In general, the CdIr, Ir and Cd groups had more apoptotic signals than the C group. Within the 12 sets of experimental results, two showed significant synergistic effects, one showed a weakly synergistic effect and nine showed additive effects. The multiple stressor effect of 100 μM Cd with ∼4.4 mGy alpha-particle radiation resulted in an additive or synergistic effect, but no antagonistic effect. The failure to identify significant synergistic effects for some sets of data, and thus their subsequent classification as additive effects, might be a result of the relatively small magnitude of the synergistic effects. The results showed that the radiation risk could be perturbed by another environmental stressor such as a heavy metal, and as such a realistic human radiation risk assessment should in general take into account the multiple stressor effects. (paper)

Production of bovine cloned embryos with donor cells frozen at a slow cooling rate in a conventional freezer (20 C)

Science.gov (United States)

Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

2009-01-01

Summary Usually, fibroblasts are frozen in dimethyl sulphoxide (DMSO, 10% v/v) at a cooling rate of 1 C/min in a low-temperature (80 C) freezer (LTF) before storage in liquid nitrogen (LN2); however, a LTF is not always available. The purpose of the present study was to evaluate apoptosis and viability of bovine fibroblasts frozen in a LTF or conventional freezer (CF; 20 C) and their subsequent ability for development to blastocyst stage after fusion with enucleated bovine oocytes. Percentages of live cells frozen in LTF (49.5%) and CF (50.6%) were similar, but significantly less than non-frozen control (88%). In both CF and LTF, percentages of live apoptotic cells exposed to LN2 after freezing were lower (4% and 5%, respectively) as compared with unexposed cells (10% and 18%, respectively). Cells frozen in a CF had fewer cell doublings/24 h (0.45) and required more days (9.1) to reach 100% confluence at the first passage (P) after thawing and plating as compared with cells frozen in a LTF (0.96 and 4.0 days, respectively). Hypoploidy at P12 was higher than at P4 in cells frozen in either a CF (37.5% vs. 19.2%) or in a LTF (30.0% vs. 15.4%). A second-generation cryo-solution reduced the incidence of necrosis (29.4%) at 0 h after thawing as compared with that of a first generation cryo-solution (DMEM + DMSO, 60.2%). The percentage of apoptosis in live cells was affected by cooling rate (CF = 1.9% vs. LFT = 0.7%). Development of bovine cloned embryos to the blastocyst stage was not affected by cooling rate or freezer type. ?? 2009 Cambridge University Press.

Toxicity potential of residual ethylene oxide on fresh or frozen embryos maintained in plastic straws.

Science.gov (United States)

Schiewe, M C; Schmidt, P M; Pontbriand, D; Wildt, D E

1988-01-01

The toxic effects of residual ethylene oxide (EtO), a frequently used gas-sterilant, on embryos either frozen for long-term purposes or stored acutely for 30 min to 9 hr in a fresh condition in 0.25-ml straw containers were evaluated. In Experiment 1, fresh embryos were frozen (using conventional technology) in straws previously aerated for 0 hr to 8 mo after EtO sterilization. With the exception of the 8-mo group in which survival and quality ratings were depressed, embryo viability was not affected significantly by short-term prefreeze and post-thaw exposure to EtO residues. Experiment 2 was conducted to analyze the influence of prefreeze exposure to EtO residues on embryo development in vitro for embryos temporarily stored in previously sterilized straws aerated for different intervals. Compared to non-EtO-sterilized control straws, the development, quality, and viability of embryos exposed to EtO-treated straws were compromised (p less than 0.05) as the aeration interval decreased and the exposure interval increased. The combined results of both experiments indicate that EtO-treated straws can be used to cryopreserve gametes efficiently, but only if the aeration interval is greater than or equal to 72 hr and the prefreeze duration of exposure is less than or equal to 3 hr.

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

DEFF Research Database (Denmark)

Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

2002-01-01

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

Proanthocyanidins Prevent High Glucose-Induced Eye Malformation by Restoring Pax6 Expression in Chick Embryo

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Rui-Rong Tan

2015-08-01

Full Text Available Gestational diabetes mellitus (GDM is one of the leading causes of offspring malformations, in which eye malformation is an important disease. It has raised demand for therapy to improve fetal outcomes. In this study, we used chick embryo to establish a GDM model to study the protective effects of proanthocyanidins on eye development. Chick embryos were exposed to high glucose (0.2 mmol/egg on embryo development day (EDD 1. Proanthocyanidins (1 and 10 nmol/egg were injected into the air sac on EDD 0. Results showed that both dosages of proanthocyanidins could prevent the eye malformation and rescue the high glucose-induced oxidative stress significantly, which the similar effects were showed in edaravone. However, proanthocyanidins could not decrease the glucose concentration of embryo eye. Moreover, the key genes regulating eye development, Pax6, was down-regulated by high glucose. Proanthocyanidins could restore the suppressed expression of Pax6. These results indicated proanthocyanidins might be a promising natural agent to prevent high glucose-induced eye malformation by restoring Pax6 expression.

Migration and growth of protoplanetary embryos. I. Convergence of embryos in protoplanetary disks

Energy Technology Data Exchange (ETDEWEB)

Zhang, Xiaojia; Lin, Douglas N. C. [Department of Astronomy and Astrophysics, University of California, Santa Cruz, CA 95064 (United States); Liu, Beibei [Kavli Institute for Astronomy and Astrophysics and Department of Astronomy, School of Physics, Peking University, Beijing 100871 (China); Li, Hui, E-mail: xzhang47@ucsc.edu [Los Alamos National Laboratory, Los Alamos, NM 87545 (United States)

2014-12-10

According to the core accretion scenario, planets form in protostellar disks through the condensation of dust, coagulation of planetesimals, and emergence of protoplanetary embryos. At a few AU in a minimum mass nebula, embryos' growth is quenched by dynamical isolation due to the depletion of planetesimals in their feeding zone. However, embryos with masses (M{sub p} ) in the range of a few Earth masses (M {sub ⊕}) migrate toward a transition radius between the inner viscously heated and outer irradiated regions of their natal disk. Their limiting isolation mass increases with the planetesimals surface density. When M{sub p} > 10 M {sub ⊕}, embryos efficiently accrete gas and evolve into cores of gas giants. We use a numerical simulation to show that despite stream line interference, convergent embryos essentially retain the strength of non-interacting embryos' Lindblad and corotation torques by their natal disks. In disks with modest surface density (or equivalently accretion rates), embryos capture each other in their mutual mean motion resonances and form a convoy of super-Earths. In more massive disks, they could overcome these resonant barriers to undergo repeated close encounters, including cohesive collisions that enable the formation of massive cores.

Increased blastocyst formation of cloned porcine embryos produced with donor cells pre-treated with Xenopus egg extract and/or digitonin

DEFF Research Database (Denmark)

Liu, Ying; Østrup, Olga; Li, Juan

2012-01-01

from Xenopus laevis eggs. In Experiment 1, fetal fibroblasts were permeabilized by digitonin, incubated in egg extract and, after re-sealing of cell membranes, cultured for 3 or 5 days before use as donor cells in handmade cloning (HMC). Controls were produced by HMC with non-treated donor cells....... The blastocyst rate for reconstructed embryos increased significantly when digitonin-permeabilized, extract-treated cells were used after 5 days of culture after re-sealing. In Experiment 2, fetal and adult fibroblasts were treated with digitonin alone before re-sealing the cell membranes, then cultured for 3...... cells after pre-treatment with permeabilization/re-sealing and Xenopus egg extract. Interestingly, we observe a similar increase in cloning efficiency by permeabilization/re-sealing of donor cells without extract treatment that seems to depend on choice of donor cell type. Thus, pre-treatment of donor...

Optimization of CRISPR/Cas9 genome editing for loss-of-function in the early chick embryo.

Science.gov (United States)

Gandhi, Shashank; Piacentino, Michael L; Vieceli, Felipe M; Bronner, Marianne E

2017-12-01

The advent of CRISPR/Cas9 has made genome editing possible in virtually any organism, including those not previously amenable to genetic manipulations. Here, we present an optimization of CRISPR/Cas9 for application to early avian embryos with improved efficiency via a three-fold strategy. First, we employed Cas9 protein flanked with two nuclear localization signal sequences for improved nuclear localization. Second, we used a modified guide RNA (gRNA) scaffold that obviates premature termination of transcription and unstable Cas9-gRNA interactions. Third, we used a chick-specific U6 promoter that yields 4-fold higher gRNA expression than the previously utilized human U6. For rapid screening of gRNAs for in vivo applications, we also generated a chicken fibroblast cell line that constitutively expresses Cas9. As proof of principle, we performed electroporation-based loss-of-function studies in the early chick embryo to knock out Pax7 and Sox10, key transcription factors with known functions in neural crest development. The results show that CRISPR/Cas9-mediated deletion causes loss of their respective proteins and transcripts, as well as predicted downstream targets. Taken together, the results reveal the utility of this optimized CRISPR/Cas9 method for targeted gene knockout in chicken embryos in a manner that is reproducible, robust and specific. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of Calcium Chloride on the Permeation of the Cryoprotectant Dimethyl Sulfoxide to Japanese Whiting Sillago japonica Embryos

Science.gov (United States)

Rahman, Sk. Mustafizur; Majhi, Sullip Kumar; Suzuki, Toru; Strussmann, Carlos Augusto; Watanabe, Manabu

Cryopreservation of fish eggs and embryos is a highly desired tool to promote aquaculture production and fisheries resource management, but it is still not technically feasible. The failure to develop successful cryopreservation protocols for fish embryos is largely attributed to poor cryoprotectant permeability. The purpose of this study was to test the effectiveness of CaCl2 to enhance cryoprotectant uptake by fish embryos. In this study, embryos (somites and tail elongation stages) of Japanese whiting Sillago japonica were exposed to 10 and 15% dimethyl sulfoxide (DMSO) in artificial sea water (ASW) or a solution of 0.125M CaCl2 in distilled water for 20 min at 24°C. The toxicity of all solutions was estimated from the hatching rates of the embryos and High Performance Liquid Chromatography was used to determine the amount of DMSO taken up during impregnation. The results showed that DMSO incorporation into the embryos was greatly (›50%) enhanced in the presence of CaCl2 compared to ASW. CaCl2 itself was not toxic to the embryos but, probably as a result of the enhanced DMSO uptake, caused decreases in survival of about 14-44% relative to ASW. Somites stage embryos were more tolerant than tail elongation ones to DMSO both as ASW and CaCl2 solutions. The use of CaCl2 as a vehicle for DMSO impregnation could be a promising aid for the successful cryopreservation of fish embryos.

Who abandons embryos after IVF?

LENUS (Irish Health Repository)

Walsh, A P H

2010-04-01

This investigation describes features of in vitro fertilisation (IVF) patients who never returned to claim their embryos following cryopreservation. Frozen embryo data were reviewed to establish communication patterns between patient and clinic; embryos were considered abandoned when 1) an IVF patient with frozen embryo\\/s stored at our facility failed to make contact with our clinic for > 2 yrs and 2) the patient could not be located after a multi-modal outreach effort was undertaken. For these patients, telephone numbers had been disconnected and no forwarding address was available. Patient, spouse and emergency family contact\\/s all escaped detection efforts despite an exhaustive public database search including death records and Internet directory portals. From 3244 IVF cycles completed from 2000 to 2008, > or = 1 embryo was frozen in 1159 cases (35.7%). Those without correspondence for > 2 yrs accounted for 292 (25.2%) patients with frozen embryos; 281 were contacted by methods including registered (signature involving abandoned embryos did not differ substantially from other patients. The goal of having a baby was achieved by 10\\/11 patients either by spontaneous conception, adoption or IVF. One patient moved away with conception status unconfirmed. The overall rate of embryo abandonment was 11\\/1159 (< 1%) in this IVF population. Pre-IVF counselling minimises, but does not totally eliminate, the problem of abandoned embryos. As the number of abandoned embryos from IVF accumulates, their fate urgently requires clarification. We propose that clinicians develop a policy consistent with relevant Irish Constitutional provisions to address this medical dilemma.

Microarray analysis of the transcriptional response to single or multiple doses of ionizing radiation in human subcutaneous fibroblasts

DEFF Research Database (Denmark)

Rødningen, Olaug Kristin; Overgaard, Jens; Alsner, Jan

2005-01-01

cell lines after various ionizing radiation (IR) schemes in order to provide information on potential targets for prevention and to suggest candidate genes for SNP association studies aimed at predicting individual risk of radiation-induced morbidity. PATIENTS AND METHODS: Thirty different human......BACKGROUND AND PURPOSE: Transcriptional profiling of fibroblasts derived from breast cancer patients might improve our understanding of subcutaneous radiation-induced fibrosis. The aim of this study was to get a comprehensive overview of the changes in gene expression in subcutaneous fibroblast...... fibroblast cell lines were included in the study, and two different radiation schemes; single dose experiments with 3.5 Gy or fractionated with 3 x 3.5 Gy. Expression analyses were performed on unexposed and exposed cells after different time points. The IR response was analyzed using the statistical method...

Effect of Radiofrequency Radiation Emitted from 2G and 3G Cell Phone on Developing Liver of Chick Embryo - A Comparative Study.

Science.gov (United States)

D'Silva, Mary Hydrina; Swer, Rijied Thompson; Anbalagan, J; Rajesh, Bhargavan

2017-07-01

The increasing scientific evidence of various health hazards on exposure of Radiofrequency Radiation (RFR) emitted from both the cell phones and base stations have caused significant media attention and public discussion in recent years. The mechanism of interaction of RF fields with developing tissues of children and fetuses may be different from that of adults due to their smaller physical size and variation in tissue electromagnetic properties. The present study may provide an insight into the basic mechanisms by which RF fields interact with developing tissues in an embryo. To evaluate the possible tissue and DNA damage in developing liver of chick embryo following chronic exposure to Ultra-High Frequency/Radiofrequency Radiation (UHF/RFR) emitted from 2G and 3G cell phone. Fertilized chick embryos were incubated in four groups. Group A-experimental group exposed to 2G radiation (60 eggs), Group B- experimental group exposed to 3G radiation (60 eggs), Group C- sham exposed control group (60 eggs) and Group D- control group (48 eggs). On completion of scheduled duration, the embryos were collected and processed for routine histological studies to check structural changes in liver. The nuclear diameter and karyorrhexis changes of hepatocytes were analysed using oculometer and square reticule respectively. The liver procured from one batch of eggs from all the four groups was subjected to alkaline comet assay technique to assess DNA damage. The results were compared using one-way ANOVA test. In our study, the exposure of developing chick embryos to 2G and 3G cell phone radiations caused structural changes in liver in the form of dilated sinusoidal spaces with haemorrhage, increased vacuolations in cytoplasm, increased nuclear diameter and karyorrhexis and significantly increased DNA damage. The chronic exposure of chick embryo liver to RFR emitted from 2G and 3G cell phone resulted in various structural changes and DNA damage. The changes were more pronounced in 3

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

Science.gov (United States)

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

Dose of recombinant FSH and oestradiol concentration on day of HCG affect embryo development kinetics

DEFF Research Database (Denmark)

Muñoz, Manuel; Cruz, María; Humaidan, Peter

2012-01-01

During follicular growth, the follicle is exposed to an almost ever-changing composition of isoforms of FSH and LH, which causes a number of different and divergent biological effects. Through a time-lapse system, embryo kinetics were examined following the use of FSH only (recombinant FSH, rFSH)...

Human dental pulp stem cells and gingival fibroblasts seeded into silk fibroin scaffolds have the same ability in attracting vessels

Directory of Open Access Journals (Sweden)

Anna eWoloszyk

2016-04-01

Full Text Available Neovascularization is one of the most important processes during tissue repair and regeneration. Current healing approaches based on the use of biomaterials combined with stem cells in critical-size bone defects fail due to the insufficient implant vascularization and integration into the host tissues. Therefore, here we studied the attraction, ingrowth, and distribution of blood vessels from the chicken embryo chorioallantoic membrane into implanted silk fibroin scaffolds seeded with either human dental pulp stem cells or human gingival fibroblasts. Perfusion capacity was evaluated by non-invasive in vivo Magnetic Resonance Imaging while the number and density of blood vessels were measured by histomorphometry. Our results demonstrate that human dental pulp stem cells and gingival fibroblasts possess equal abilities in attracting vessels within silk fibroin scaffolds. Additionally, the prolonged in vitro pre-incubation period of these two cell populations favors the homogeneous distribution of vessels within silk fibroin scaffolds, which further improves implant survival and guarantees successful healing and regeneration.

The hallmarks of fibroblast ageing.

Science.gov (United States)

Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

2014-06-01

Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

International Nuclear Information System (INIS)

He Xiaoqing; Chen Rui; Yang Ping; Li Aiping; Zhou Jianwei; Liu Qizhan

2007-01-01

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 μM) arsenite but inhibited at higher concentrations (5 and 10 μM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 μM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 μM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 μM SP600125 or ERK1/2 by 100 μM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process

Developmental toxicity evaluation of three hexabromocyclododecane diastereoisomers on zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Du Miaomiao [Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Graduate University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang Dandan; Yan Changzhou [Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China); Zhang Xian, E-mail: xzhang@iue.ac.cn [Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021 (China)

2012-05-15

Structural dissimilarities of hexabromocyclododecane diastereoisomers could raise substantial differences in physicochemical, biological and toxicological properties. In order to fully assess the environmental safety and health risk of hexabromocyclododecanes (HBCDs), zebrafish embryos were used to evaluate the developmental toxicity of individual HBCD diastereoisomers ({alpha}-HBCD, {beta}-HBCD and {gamma}-HBCD). Four-hour post-fertilization (hpf) zebrafish embryos were exposed to different concentrations of HBCD diastereoisomers (0, 0.01, 0.1 and 1.0 mg/l) until 120 hpf. The results showed that exposure to HBCDs can affect the development of zebrafish embryos/larvae in a dose-dependent and diastereoselective manner. The diastereoisomers {alpha}-, {beta}- and {gamma}-HBCD at 0.01 mg/l had little effect on the development of zebrafish embryos except that exposure to 0.01 mg/l {gamma}-HBCD significantly delayed hatching (P < 0.05). At 0.1 mg/l, {alpha}-HBCD resulted in depressed heart rate of larvae (96 hpf) and delayed hatching, whereas {beta}- and {gamma}-HBCD both caused significant hatching delay and growth inhibition (P < 0.05). In addition, a remarkable and significant increase in mortality and malformation rate was noted at 0.1 mg/l {gamma}-HBCD exposure groups (P < 0.05). At 1.0 mg/l, {alpha}-, {beta}- and {gamma}-HBCD significantly affected all of the endpoints monitored (P < 0.05). Additionally, HBCD diastereoisomers could induce the generation of reactive oxygen species (ROS) and the activities of caspase-3 and caspase-9 in a dose-dependent manner. The results indicated that HBCD diastereoisomers could cause developmental toxicity to zebrafish embryos through inducing apoptosis by ROS formation. The overall results showed a good agreement confirming that the order of developmental toxicity of HBCD diastereoisomers in zebrafish is {gamma}-HBCD > {beta}-HBCD > {alpha}-HBCD.

Neutron fluence-to-dose conversion coefficients for embryo and fetus

International Nuclear Information System (INIS)

Chen, J.; Meyerhof, D.; Vlahovich, S.

2004-01-01

A problem of concern in radiation protection is the exposure of pregnant women to ionising radiation, because of the high radiosensitivity of the embryo and fetus. External neutron exposure is of concern when pregnant women travel by aeroplane. Dose assessments for neutrons frequently rely on fluence-to-dose conversion coefficients. While neutron fluence-to-dose conversion coefficients for adults are recommended in International Commission on Radiological Protection publications and International Commission on Radiological Units and Measurements reports, conversion coefficients for embryos and fetuses are not given in the publications. This study undertakes Monte Carlo calculations to determine the mean absorbed doses to the embryo and fetus when the mother is exposed to neutron fields. A new set of mathematical models for the embryo and fetus has been developed at Health Canada and is used together with mathematical phantoms of a pregnant female developed at Oak Ridge National Laboratory. Monoenergetic neutrons from 1 eV to 10 MeV are considered in this study. The irradiation geometries include antero-posterior (AP), postero-anterior (PA), lateral (LAT), rotational (ROT) and isotropic (ISO) geometries. At each of these standard irradiation geometries, absorbed doses to the fetal brain and body are calculated; for the embryo at 8 weeks and the fetus at 3, 6 or 9 months. Neutron fluence-to-absorbed dose conversion coefficients are derived for the four age groups. Neutron fluence-to-equivalent dose conversion coefficients are given for the AP irradiations which yield the highest radiation dose to the fetal body in the neutron energy range considered here. The results indicate that for neutrons <10 MeV more protection should be given to pregnant women in the first trimester due to the higher absorbed dose per unit neutron fluence to the fetus. (authors)

Neutron fluence-to-dose conversion coefficients for embryo and fetus.

Science.gov (United States)

Chen, Jing; Meyerhof, Dorothy; Vlahovich, Slavica

2004-01-01

A problem of concern in radiation protection is the exposure of pregnant women to ionising radiation, because of the high radiosensitivity of the embryo and fetus. External neutron exposure is of concern when pregnant women travel by aeroplane. Dose assessments for neutrons frequently rely on fluence-to-dose conversion coefficients. While neutron fluence-to-dose conversion coefficients for adults are recommended in International Commission on Radiological Protection publications and International Commission on Radiological Units and Measurements reports, conversion coefficients for embryos and fetuses are not given in the publications. This study undertakes Monte Carlo calculations to determine the mean absorbed doses to the embryo and fetus when the mother is exposed to neutron fields. A new set of mathematical models for the embryo and fetus has been developed at Health Canada and is used together with mathematical phantoms of a pregnant female developed at Oak Ridge National Laboratory. Monoenergetic neutrons from 1 eV to 10 MeV are considered in this study. The irradiation geometries include antero-posterior (AP), postero-anterior (PA), lateral (LAT), rotational (ROT) and isotropic (ISO) geometries. At each of these standard irradiation geometries, absorbed doses to the fetal brain and body are calculated; for the embryo at 8 weeks and the fetus at 3, 6 or 9 months. Neutron fluence-to-absorbed dose conversion coefficients are derived for the four age groups. Neutron fluence-to-equivalent dose conversion coefficients are given for the AP irradiations which yield the highest radiation dose to the fetal body in the neutron energy range considered here. The results indicate that for neutrons <10 MeV more protection should be given to pregnant women in the first trimester due to the higher absorbed dose per unit neutron fluence to the fetus.

Ethanol disrupts chondrification of the neurocranial cartilages in medaka embryos without affecting aldehyde dehydrogenase 1A2 (Aldh1A2) promoter methylation

Science.gov (United States)

Hu, Yuhui; Willett, Kristine L.; Khan, Ikhlas A.; Scheffler, Brian E.; Dasmahapatra, Asok K.

2009-01-01

Medaka (Oryzias latipes) embryos at different developmental stages were exposed to ethanol for 48 h, then allowed to hatch. Teratogenic effects were evaluated in hatchlings after examining chondrocranial cartilage deformities. Ethanol disrupted cartilage development in medaka in a dose and developmental stage-specific manner. Compared to controls, the linear length of the neurocranium and other cartilages were reduced in ethanol-treated groups. Moreover, the chondrification in cartilages, specifically trabeculae and polar cartilages, were inhibited by ethanol. To understand the mechanism of ethanol teratogenesis, NAD+: NADH status during embryogenesis and the methylation pattern of Aldh1A2 promoter in whole embryos and adult tissues (brain, eye, heart and liver) were analyzed. Embryos 6 dpf had higher NAD+ than embryos 0 or 2 dpf. Ethanol (200 or 400 mM) was able to reduce NAD+ content in 2 and 6 dpf embryos. However, in both cases reductions were not significantly different from the controls. Moreover, no significant difference in either NADH content or in NAD+: NADH status of the ethanol-treated embryos, with regard to controls, was observed. The promoter of Aldh1A2 contains 31 CpG dinucleotides (-705 to +154, ATG = +1); none of which were methylated. Compared to controls, embryonic ethanol exposure (100 and 400 mM) was unable to alter Aldh1A2 promoter methylation in embryos or in the tissues of adults (breeding) developmentally exposed to ethanol (300 mM, 48 hpf). From these data we conclude that ethanol teratogenesis in medaka does not induce alteration in the methylation pattern of Aldh1A2 promoter, but does change cartilage development. PMID:19651241

Cytotoxicity Evaluation of Two Bis-Acryl Composite Resins Using Human Gingival Fibroblasts.

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Gonçalves, Fabiano Palmeira; Alves, Gutemberg; Guimarães, Vladi Oliveira; Gallito, Marco Antônio; Oliveira, Felipe; Scelza, Míriam Zaccaro

2016-01-01

Bis-acryl resins are used for temporary dental restorations and have shown advantages over other materials. The aim of this work was to evaluate the in vitro cytotoxicity of two bis-acryl composite resins (Protemp 4 and Luxatemp Star), obtained at 1, 7 and 40 days after mixing the resin components, using a standardized assay employing human primary cells closely related to oral tissues. Human gingival fibroblast cell cultures were exposed for 24 h to either bis-acryl composite resins, polystyrene beads (negative control) and latex (positive control) extracts obtained after incubation by the different periods, at 37 °C under 5% CO2. Cell viability was evaluated using a multiparametric procedure involving sequential assessment (using the same cells) of mitochondrial activity (XTT assay), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). The cells exposed to the resin extracts showed cell viability indexes exceeding 75% after 24 h. Even when cells were exposed to extracts prepared with longer conditioning times, the bis-acryl composite resins showed no significant cytotoxic effects (p>0.05), compared to the control group or in relation to the first 24 h of contact with the products. There were no differences among the results obtained for the bis-acryl composite resins evaluated 24 h, 7 days and 40 days after mixing. It may be concluded that the bis-acryl resins Protemp 4 and Luxatemp Star were cytocompatible with human gingival fibroblasts, suggesting that both materials are suitable for use in contact with human tissues.

Production of Pigs by Hand-Made Cloning Using Mesenchymal Stem Cells and Fibroblasts.

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Yang, Zhenzhen; Vajta, Gábor; Xu, Ying; Luan, Jing; Lin, Mufei; Liu, Cong; Tian, Jianing; Dou, Hongwei; Li, Yong; Liu, Tianbin; Zhang, Yijie; Li, Lin; Yang, Wenxian; Bolund, Lars; Yang, Huanming; Du, Yutao

2016-08-01

Mesenchymal stem cells (MSCs) exhibited self-renewal and less differentiation, making the MSCs promising candidates for adult somatic cell nuclear transfer (SCNT). In this article, we tried to produce genome identical pigs through hand-made cloning (HMC), with MSCs and adult skin fibroblasts as donor cells. MSCs were derived from either adipose tissue or peripheral blood (aMSCs and bMSCs, respectively). MSCs usually showed the expression pattern of CD29, CD73, CD90, and CD105 together with lack of expression of the hematopoietic markers CD34and CD45. Flow cytometry results demonstrated high expression of CD29 and CD90 in both MSC lines, while CD73, CD34, and CD45 expression were not detected. In contrary, in reverse transcription-polymerase chain reaction (RT-PCR) analysis, CD73 and CD34 were detected indicating that human antibodies CD73 and CD34 were not suitable to identify porcine cell surface markers and porcine MSC cellular surface markers of CD34 might be different from other species. MSCs also had potential to differentiate successfully into chondrocytes, osteoblasts, and adipocytes. After HMC, embryos reconstructed with aMSCs had higher blastocyst rate on day 5 and 6 than those reconstructed with bMSCs and fibroblasts (29.6% ± 1.3% and 41.1% ± 1.4% for aMSCs vs. 23.9% ± 1.2% and 35.5% ± 1.6% for bMSCs and 22.1% ± 0.9% and 33.3% ± 1.1% for fibroblasts, respectively). Live birth rate per transferred blastocyst achieved with bMSCs (1.59%) was the highest among the three groups. This article was the first report to compare the efficiency among bMSCs, aMSCs, and fibroblasts for boar cloning, which offered a realistic perspective to use the HMC technology for commercial breeding.

Transcriptional responses of Acropora hyacinthus embryo under the benzo(a)pyrene stress by deep sequencing.

Science.gov (United States)

Xiao, Rong; Zhou, Hailong; Chen, Chien-Min; Cheng, Huamin; Li, Hongwu; Xie, Jia; Zhao, Hongwei; Han, Qian; Diao, Xiaoping

2018-04-24

Coral embryos are a critical and sensitive period for the early growth and development of coral. Benzo(a)pyrene (BaP) is widely distributed in the ocean and has strong toxicity, but there is little information on the toxic effects to coral embryos exposed to this widespread environmental contaminant. Thus, in this study, we utilized the Illumina Hiseq™ 4000 platform to explore the gene response of Acropora hyacinthus embryos under the BaP stress. A total of 130,042 Unigenes were obtained and analyzed, and approximately 37.67% of those matched with sequences from four different species. In total, 2606 Unigenes were up-regulated, and 3872 Unigenes were down-regulated. After Gene Ontology (GO) annotation, the results show that the "cellular process" and "metabolic process" were leading in the category of biological processes, which the "binding" and "catalytic activity" were the most abundant subcategories in molecular function. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the most differentially expressed genes (DEGs) were enriched, as well as down-regulated in the pathways of oxidative phosphorylation, metabolism of xenobiotics, immune-related genes, apoptosis and human disease genes. At the same time, 388,197 of Single-nucleotide Polymorphisms (SNPs) and 6164 of Simple Sequence Repeats (SSRs) were obtained, which can be served as the richer and more valuable SSRs molecular markers in the future. The results of this study can help to better understand the toxicological mechanism of coral embryo exposed to BaP, and it is also essential for the protection and restoration of coral reef ecosystem in the future. Copyright © 2018 Elsevier Ltd. All rights reserved.

The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

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Paweł P. Antończak

2017-07-01

Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

Effects of atrazine on embryos, larvae, and adults of anuran amphibians.

Science.gov (United States)

Allran, J W; Karasov, W H

2001-04-01

We examined the effects of atrazine (0-20 mg/L) on embryos, larvae, and adult anuran amphibian species in the laboratory. Atrazine treatments did not affect hatchability of embryos or 96-h posthatch mortality of larvae of Rana pipiens, Rana sylvatica, or Bufo americanus. Furthermore, atrazine had no effect on swimming speed (measured for R. pipiens only). However, there was a dose-dependent increase in deformed larvae of all three species with increasing atrazine concentration. In adult R. pipiens, atrazine increased buccal and thoracic ventilation, indicating respiratory distress. However, because atrazine had no affect on hemoglobin, this respiratory distress was probably not indicative of reduced oxygen-carrying capacity of the blood. Frogs exposed to the highest atrazine concentration stopped eating immediately after treatment began and did not eat during the 14-d experiment. However, no decreases in mass were measured even for frogs that were not eating, probably because of compensatory fluid gain from edema. Atrazine concentrations found to be deleterious to amphibian embryos and adults are considerably higher than concentrations currently found in surface waters in North America. Therefore, direct toxicity of atrazine is probably not a significant factor in recent amphibian declines.

Fibroblast growth factor receptors in breast cancer.

Science.gov (United States)

Wang, Shuwei; Ding, Zhongyang

2017-05-01

Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

Frozen-Thawed Embryo Transfer Cycles Have a Lower Incidence of Ectopic Pregnancy Compared With Fresh Embryo Transfer Cycles.

Science.gov (United States)

Zhang, Xinyu; Ma, Caihong; Wu, Zhangxin; Tao, Liyuan; Li, Rong; Liu, Ping; Qiao, Jie

2017-01-01

To evaluate the risk of ectopic pregnancy of embryo transfer. A retrospective cohort study on the incidence of ectopic pregnancy in fresh and frozen-thawed embryo transfer cycles from January 1 st , 2010, to January 1 st , 2015. Infertile women undergoing frozen-thawed transfer cycles or fresh transfer cycles. In-vitro fertilization, fresh embryo transfer, frozen-thawed embryo transfer, ectopic pregnancy. Ectopic pregnancy rate and clinical pregnancy rate. A total of 69 756 in vitro fertilization-embryo transfer cycles from 2010 to 2015 were analyzed, including 45 960 (65.9%) fresh and 23 796 (34.1%) frozen-thawed embryo transfer cycles. The clinical pregnancy rate per embryo transfer was slightly lower in fresh embryo transfer cycles compared with frozen-thawed embryo transfer cycles (40.8% vs 43.1%, P cycles, blastocyst transfer shows a significantly lower incidence of ectopic pregnancy (0.8% vs 1.8%, P = .002) in comparison with day 3 cleavage embryo transfer. The risk of ectopic pregnancy is lower in frozen-thawed embryo transfer cycles than fresh embryo transfer cycles, and blastocyst transfer could further decrease the ectopic pregnancy rate in frozen-thawed embryo transfer cycles.

Ontogeny of salinity tolerance and hyper-osmoregulation by embryos of the intertidal crabs Hemigrapsus edwardsii and Hemigrapsus crenulatus (Decapoda, Grapsidae): survival of acute hyposaline exposure.

Science.gov (United States)

Taylor, H H; Seneviratna, Deepani

2005-04-01

The adults of Hemigrapsus edwardsii and Hemigrapsus crenulatus are euryhaline crabs and strong hyper-osmoregulators. Their embryos are carried externally attached to the abdominal pleopods of female crabs, where they are exposed to temporal and spatial changes in salinity associated with their intertidal and estuarine habitats. Although embryos lack the branchial and excretory organs responsible for adult osmoregulation, post-gastrula embryos were highly tolerant of exposure to hypo-osmotic sea water. Detached eggs (embryos+envelopes), of both species, at all developmental stages between gastrulation and hatching, exhibited 80-100% survival for periods up to 96 h in sea water (osmolality, 1050 mmol kg(-1)) and in dilutions to 50%, 10%, and 1%. Cleavage stages were less tolerant of dilution; H. edwardsii, <50% survived 24 h in 10% sea water; H. crenulatus <50% survived 6 h in 10% sea water. Post-gastrulation stages strongly hyper-osmoregulated but cleavage stages were hyper-osmoconformers (maintaining internal osmolality approximately 150 mmol kg(-1) above external). Osmoregulatory capacity was reduced just prior hatching, particularly in H. crenulatus, although salinity tolerance remained high. Gastrulation therefore marks a critical stage in the ontogeny of osmoregulation and salinity tolerance. Total Na+/K(+)-ATPase activity increased greatly during embryogenesis of H. crenulatus (undetectable in blastulae; gastrulae 0.31+/-0.05 pmol P(i) embryo(-1) min(-1); pre-hatching 16.4+/-1.0 pmol P(i) embryo(-1) min(-1)). Na+/K(+)-ATPase activity increased in embryos exposed to dilute sea water for 24 h implicating regulation of this transporter in a short-term acclimation response.

In situ exposure of herring embryos in Prince William Sound two years after the Exxon Valdez oil spill

International Nuclear Information System (INIS)

Kocan, R.; Brown, E.; Baker, T.

1995-01-01

In order to evaluate long-term residual toxicity, artificially spawned Pacific herring (Clupea pallasl) embryos were deployed at 5 oiled and 5 unoiled sites in Prince William Sound two years after the Exxon Valdez oil spill. Embryos were exposed at 1.5 m and 4.5 m below mean low water for 8--10 d post fertilization. The embryos were then retrieved and transported to the laboratory for hatching and evaluation under controlled conditions, Endpoints were (1) embryo survival, (2) live hatch, (3) deformed larvae and (4) larval dry weight. The oiled sites produced significantly (P < 0.01) more deformed larvae (63.3%) than did the unoiled sites (51.3%), but there was a lower hatching success at the unoiled sites which resulted in no overall difference in normal live larvae produced between oiled and unoiled sites. The mean dry weight of newly hatched larvae from the oiled sites (78 microg/larva) was significantly lower than those from the unoiled sites (97 microg/larva) at all depths (P < 0.01). Increased larval deformities and reduced hatching weight is consistent with what has been reported by several investigators for herring larvae experimentally and naturally exposed to petroleum hydrocarbons. Based on the data, there appeared to be differences between the previously oiled and unoiled sites relative to herring development, but it is not clear whether this was due to residual oil effects or parental effects

The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Zeng, Cheng [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Sun, Hong [Hubei Maternal and Child Health Hospital, Wuhan 430070 (China); Xie, Ping [Donghu Experimental Station of Lake Ecosystems, State Key Laboratory for Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072 (China); Wang, Jianghua; Zhang, Guirong; Chen, Nan [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Yan, Wei, E-mail: Yanwei75126@163.com [Institute of Agricultural Quality Standards and Testing Technology, Hubei Academy of Agricultural Sciences, Wuhan 430064 (China); Li, Guangyu, E-mail: ligy2001@163.com [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070 (China)

2014-04-01

Highlights: • MCLR-induced apoptosis in the heart of developing embryos leads to the growth delay in zebrafish. • MCLR-triggered apoptosis might be induced by ROS. • P53–Bax–Bcl-2 and caspase-dependent apoptotic pathway contribute greatly to MCLR-induced apoptosis. Abstract: We previously demonstrated that cyanobacteria-derived microcystin–leucine–arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L⁻¹ for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L⁻¹ MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53–Bax–Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos.

Polycyclic aromatic hydrocarbons affect survival and development of common snapping turtle (Chelydra serpentina) embryos and hatchlings.

Science.gov (United States)

Van Meter, Robin J; Spotila, James R; Avery, Harold W

2006-08-01

Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds found in the John Heinz National Wildlife Refuge in Philadelphia, Pennsylvania. We assessed the impact of PAHs and crude oil on snapping turtle development and behavior by exposing snapping turtle eggs from the Refuge and from three clean reference sites to individual PAHs or a crude oil mixture at stage 9 of embryonic development. Exposure to PAHs had a significant effect on survival rates in embryos from one clean reference site, but not in embryos from the other sites. There was a positive linear relationship between level of exposure to PAHs and severity of deformities in embryos collected from two of the clean reference sites. Neither righting response nor upper temperature tolerance (critical thermal maximum, CTM) of snapping turtle hatchlings with no or minor deformities was significantly affected by exposure to PAHs.

Growth-arrest-specific protein 2 inhibits cell division in Xenopus embryos.

Directory of Open Access Journals (Sweden)

Tong Zhang

Full Text Available Growth-arrest-specific 2 gene was originally identified in murine fibroblasts under growth arrest conditions. Furthermore, serum stimulation of quiescent, non-dividing cells leads to the down-regulation of gas2 and results in re-entry into the cell cycle. Cytoskeleton rearrangements are critical for cell cycle progression and cell division and the Gas2 protein has been shown to co-localize with actin and microtubules in interphase mammalian cells. Despite these findings, direct evidence supporting a role for Gas2 in the mechanism of cell division has not been reported.To determine whether the Gas2 protein plays a role in cell division, we over-expressed the full-length Gas2 protein and Gas2 truncations containing either the actin-binding CH domain or the tubulin-binding Gas2 domain in Xenopus laevis embryos. We found that both the full-length Gas2 protein and the Gas2 domain, but not the CH domain, inhibited cell division and resulted in multinucleated cells. The observation that Gas2 domain alone can arrest cell division suggests that Gas2 function is mediated by microtubule binding. Gas2 co-localized with microtubules at the cell cortex of Gas2-injected Xenopus embryos using cryo-confocal microscopy and co-sedimented with microtubules in cytoskeleton co-sedimentation assays. To investigate the mechanism of Gas2-induced cell division arrest, we showed, using a wound-induced contractile array assay, that Gas2 stabilized microtubules. Finally, electron microscopy studies demonstrated that Gas2 bundled microtubules into higher-order structures.Our experiments show that Gas2 inhibits cell division in Xenopus embryos. We propose that Gas2 function is mediated by binding and bundling microtubules, leading to cell division arrest.

Transcriptional response of zebrafish embryos exposed to neurotoxic compounds reveals a muscle activity dependent hspb11 expression.

Directory of Open Access Journals (Sweden)

Nils Klüver

Full Text Available Acetylcholinesterase (AChE inhibitors are widely used as pesticides and drugs. Their primary effect is the overstimulation of cholinergic receptors which results in an improper muscular function. During vertebrate embryonic development nerve activity and intracellular downstream events are critical for the regulation of muscle fiber formation. Whether AChE inhibitors and related neurotoxic compounds also provoke specific changes in gene transcription patterns during vertebrate development that allow them to establish a mechanistic link useful for identification of developmental toxicity pathways has, however, yet not been investigated. Therefore we examined the transcriptomic response of a known AChE inhibitor, the organophosphate azinphos-methyl (APM, in zebrafish embryos and compared the response with two non-AChE inhibiting unspecific control compounds, 1,4-dimethoxybenzene (DMB and 2,4-dinitrophenol (DNP. A highly specific cluster of APM induced gene transcripts was identified and a subset of strongly regulated genes was analyzed in more detail. The small heat shock protein hspb11 was found to be the most sensitive induced gene in response to AChE inhibitors. Comparison of expression in wildtype, ache and sop(fixe mutant embryos revealed that hspb11 expression was dependent on the nicotinic acetylcholine receptor (nAChR activity. Furthermore, modulators of intracellular calcium levels within the whole embryo led to a transcriptional up-regulation of hspb11 which suggests that elevated intracellular calcium levels may regulate the expression of this gene. During early zebrafish development, hspb11 was specifically expressed in muscle pioneer cells and Hspb11 morpholino-knockdown resulted in effects on slow muscle myosin organization. Our findings imply that a comparative toxicogenomic approach and functional analysis can lead to the identification of molecular mechanisms and specific marker genes for potential neurotoxic compounds.

Effect of chloramphenicol on sister chromatid exchange in bovine fibroblasts.

Science.gov (United States)

Arruga, M V; Catalan, J; Moreno, C

1992-03-01

The genotoxic potential of different chloramphenicol concentrations (5, 20, 40 and 60 micrograms ml-1) was investigated in bovine fibroblast primary lines by sister chromatid exchange assay. Chloramphenicol acted for long enough to ensure similar effects to persistent storage in the kidney. In this experiment 10 micrograms ml-1 of 5-bromodeoxyuridine was added for 60 hours for all doses of chloramphenicol and to the control. When the tissue culture cells were exposed to increasing doses, increased numbers of sister chromatid exchanges developed. Differences were significantly different to the control.

Combination effects of AHR agonists and Wnt/β-catenin modulators in zebrafish embryos: Implications for physiological and toxicological AHR functions

Energy Technology Data Exchange (ETDEWEB)

Wincent, Emma [Department of Environmental Toxicology, Uppsala University, 75236 Uppsala (Sweden); Institute of Environmental Medicine, Karolinska Institutet, 17177 Stockholm (Sweden); Stegeman, John J. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543-1050 (United States); Jönsson, Maria E., E-mail: maria.jonsson@ebc.uu.se [Department of Environmental Toxicology, Uppsala University, 75236 Uppsala (Sweden)

2015-04-15

Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examined phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.

Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model

Science.gov (United States)

Hu, Yu-Lan; Qi, Wang; Han, Feng; Shao, Jian-Zhong; Gao, Jian-Qing

2011-01-01

Background Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In the present study, the zebrafish model was used for a safety evaluation of this nanocarrier. Methods Chitosan nanoparticles with two particle sizes were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles of different concentrations were incubated with zebrafish embryos, and ZnO nanoparticles were used as the positive control. Results Embryo exposure to chitosan nanoparticles and ZnO nanoparticles resulted in a decreased hatching rate and increased mortality, which was concentration-dependent. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Furthermore, embryos exposed to chitosan nanoparticles showed an increased rate of cell death, high expression of reactive oxygen species, as well as overexpression of heat shock protein 70, indicating that chitosan nanoparticles can cause physiological stress in zebrafish. The results also suggest that the toxicity of biodegradable nanocarriers such as chitosan nanoparticles must be addressed, especially considering the in vivo distribution of these nanoscaled particles. Conclusion Our results add new insights into the potential toxicity of nanoparticles produced by biodegradable materials, and may help us to understand better the nanotoxicity of drug delivery carriers. PMID:22267920

A brief introduction of ICRP Publication 49: Developmental effects of irradiation on the brain of the embryo and fetus

International Nuclear Information System (INIS)

Kusama, Tomoko

1988-01-01

ICRP established a task group within its Committee 1 to carry out studies on the effects of irradiation on the central nerve system of embryos and fetuses. The present article summarizes the study results presented in the report, named ICRP Publication 49, published by the task group. Publication 49 consists of seven chapters dealing with the introduction, development of brain and auxiliary organs of embryo primates, retarded development of central nerve system, ionizing radiation as factor in teratogenesis of central nerve system, maximum susceptibility period, risk estimation for human being, and necessity of research. Radiation may cause either organogenetic or histological disturbances depending on the developmental stage of the brain. Results of animal tests can be applied to studies on the morphogenetic disturbances in human beings. Data on embryos and fetuses that received radiation in Hiroshima or Nagasaki are currently used for the estimation of the risk of disturbance in the brain of human embryos and fetuses. Risk estimation for the brain of human embryo exposed to radiation is discussed. (Nogami, K.)

Novel transcriptome assembly and comparative toxicity pathway analysis in mahi-mahi (Coryphaena hippurus) embryos and larvae exposed to Deepwater Horizon oil

Science.gov (United States)

Xu, Elvis Genbo; Mager, Edward M.; Grosell, Martin; Hazard, E. Starr; Hardiman, Gary; Schlenk, Daniel

2017-03-01

The impacts of Deepwater Horizon (DWH) oil on morphology and function during embryonic development have been documented for a number of fish species, including the economically and ecologically important pelagic species, mahi-mahi (Coryphaena hippurus). However, further investigations on molecular events and pathways responsible for developmental toxicity have been largely restricted due to the limited molecular data available for this species. We sought to establish the de novo transcriptomic database from the embryos and larvae of mahi-mahi exposed to water accommodated fractions (HEWAFs) of two DWH oil types (weathered and source oil), in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses. By high throughput sequencing (HTS), we obtained the first de novo transcriptome of mahi-mahi, with 60,842 assembled transcripts and 30,518 BLAST hits. Among them, 2,345 genes were significantly regulated in 96hpf larvae after exposure to weathered oil. With comparative analysis to a reference-transcriptome-guided approach on gene ontology and tox-pathways, we confirmed the novel approach effective for exploring tox-pathways in non-model species, and also identified a list of co-expressed genes as potential biomarkers which will provide information for the construction of an Adverse Outcome Pathway which could be useful in Ecological Risk Assessments.

Surgical manipulation of mammalian embryos in vitro.

Science.gov (United States)

Naruse, I; Keino, H; Taniguchi, M

1997-04-01

Whole-embryo culture systems are useful in the fields of not only embryology but also teratology, toxicology, pharmacology, and physiology. Of the many advantages of whole-embryo culture, we focus here on the surgical manipulation of mammalian embryos. Whole-embryo culture allows us to manipulate mammalian embryos, similarly to fish, amphibian and avian embryos. Many surgical experiments have been performed in mammalian embryos in vitro. Such surgical manipulation alters the destiny of morphogenesis of the embryos and can answer many questions concerning developmental issues. As an example of surgical manipulation using whole-embryo culture systems, one of our experiments is described. Microsurgical electrocauterization of the deep preaxial mesodermal programmed cell death zone (fpp) in the footplate prevented the manifestation of polydactyly in genetic polydactyly mouse embryos (Pdn/Pdn), in which fpp was abolished.

Nuclear and nuclear reprogramming during the first cell cycle in bovine nuclear transfer embryos

DEFF Research Database (Denmark)

Østrup, Olga; Petrovicova, Ida; Strejcek, Frantisek

2009-01-01

Abstract The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed......, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially...... restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of development....

Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

Science.gov (United States)

Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

2015-12-01

Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in the ICM occurred, the number of eGFP-positive cells in this compartment was 8.3 ± 2.3 (mean ± standard error of the mean). We conclude that PHA is advantageous for the formation of aggregation chimeras, but the approach tested in the present study with only two donor blastomeres and two host embryos did not result in multiplication of genetically valuable donor embryos. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

Energy Technology Data Exchange (ETDEWEB)

Zhen, Zhen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Luthringer, Bérengère, E-mail: berengere.luthringer@hzg.de [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Yang, Li [College of Engineering, Peking University, Beijing 100871 (China); Xi, Tingfei, E-mail: xitingfei@pku.edu.cn [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 (China); Zheng, Yufeng [Shenzhen Institute, Peking University, Shenzhen 518057 (China); College of Engineering, Peking University, Beijing 100871 (China); Feyerabend, Frank; Willumeit, Regine [Institute of Material Research, Helmholtz-Zentrum Geesthacht, Hamburg 21502 (Germany); Lai, Chen [Shenzhen Institute, Peking University, Shenzhen 518057 (China); Ge, Zigang [College of Engineering, Peking University, Beijing 100871 (China)

2016-12-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

Proteomic profile of mouse fibroblasts exposed to pure magnesium extract

International Nuclear Information System (INIS)

Zhen, Zhen; Luthringer, Bérengère; Yang, Li; Xi, Tingfei; Zheng, Yufeng; Feyerabend, Frank; Willumeit, Regine; Lai, Chen; Ge, Zigang

2016-01-01

Magnesium and its alloys gain wide attention as degradable biomaterials. In order to reveal the molecular mechanism of the influence of biodegradable magnesium on cells, proteomics analysis was performed in this work. After mouse fibroblasts (L929) were cultured with or without Mg degradation products (Mg-extract) for 8, 24, and 48 h, changes in protein expression profiles were obtained using isobaric tags for relative and absolute quantitation (iTRAQ) coupled two dimensional liquid chromatography-tandem mass spectrometry (2D LC MS/MS). A total of 867 proteins were identified (relying on at least two peptides). Compared to the control group, 205, 282, and 217 regulated proteins were identified at 8, 24, and 48 h, respectively. 65 common proteins were up or down- regulated within all the three time points, which were involved in various physiological and metabolic activities. Consistent with viability, proliferation, and cell cycle analysis, stimulated energy metabolism as well as protein synthesis pathways were discussed, indicating a possible effect of Mg-extract on L929 proliferation. Furthermore, endocytosis and focal adhesion processes were also discussed. This proteomics study uncovers early cellular mechanisms triggered by Mg degradation products and highlights the cytocompatibility of biodegradable metallic materials for biomedical applications such as stents or orthopaedic implants. - Highlights: • First proteomic analysis of bioeffect mechanism caused by biodegradable Mg • Totally 867 proteins were identified by iTRAQ LC-MS/MS in this work. • 65 proteins were focused on because they were regulated within all the culture time. • The 65 proteins were associated with diverse biological processes. • Cell proliferation mechanism in Mg extract was investigated.

Ovarian stimulation and embryo quality

NARCIS (Netherlands)

Baart, Esther; Macklon, Nick S.; Fauser, Bart J. C. M.

To Study the effects of different ovarian stimulation approaches on oocyte and embryo quality, it is imperative to assess embryo quality with a reliable and objective method. Embryos rated as high quality by standardized morphological assessment are associated with higher implantation and pregnancy

Survival of embryo irradiated with gamma rays by embryo culture in Brassica pekinensis Rupr

International Nuclear Information System (INIS)

Moue, T.

1984-01-01

The effect of irradiation on the survival rates and embryonic development of Brassica pekinensis RUPR. (Varieties; Kashin, Kohai 65 nichi and kairyochitose) was investigated. The purpose of this study was to seek ways of increasing the survival rates of embryos such as B.oleracea obtained through embryo culture techniques after irradiation doses affecting seed fertility and germination, for the purpose of increasing mutation rates. Embryos at different developmental stages ranging from the globular to the early heart stages were irradiated with 20 KR of gamma rays at the daily rate 0L 20 KR or 10 KR (Fig.1 and Table 1). The embryos were excised from ovules 4 to 10 days after irradiation and cultured on White's medium. The shooting and rooting rates on the 34th day of culture were higher at the dose of 10 KR/day than 20 KR/day and were lower when the materials were irradiated at the young embryonic stage (Table 3). Varietal differences in the shooting and rooting rates were also observed. The irradiated embryos survived mainly in the state of callus. It was concluded that the embryo culture technique was successful when applied to irradiated embryos excised at the young embryonic stage and that the technique affected B.pekinensis less than B.oleracea

Tumor necrosis factor alpha inhibits in vitro bovine embryo development through a prostaglandin mediated mechanism

Directory of Open Access Journals (Sweden)

Jackson Lauren R

2012-03-01

Full Text Available Abstract Mastitis or other infectious diseases have been related to reduced fertility in cattle. Inflammatory cytokines such as tumor necrosis factor α (TNFα are released in response to infection and may have negative effects on embryo development. In the current study the effect of exposure to TNFα on the development of in vitro fertilized bovine embryos was examined. Indomethacin, a prostaglandin synthesis inhibitor, was used to determine if blockade of prostaglandin synthesis would alter the effects of TNFα. Ovaries were obtained from a local abattoir and immature COC were isolated from 2-10 mm follicles, in vitro matured and fertilized. After fertilization, groups of presumptive zygotes were randomly placed into either control development medium, medium containing 25 ng/mL TNFα or medium containing 25 ng/mL TNFα plus 1 μg/mL indomethacin. The proportion of blastocysts formed was assessed at day 7 of culture. Fewer embryos exposed to TNFα alone reached the blastocyst stage (17.5 ± 2.4%, P

Impacts of different exposure scenarios on transcript abundances in Danio rerio embryos when investigating the toxicological burden of riverine sediments.

Directory of Open Access Journals (Sweden)

Kerstin Bluhm

Full Text Available PURPOSE: Recently, a proof-of-concept study revealed the suitability of transcriptome analyses to obtain and assess changes in the abundance of transcripts in zebrafish (Danio rerio embryos after exposure to organic sediment extracts. The present study investigated changes in the transcript abundance in zebrafish embryos exposed to whole sediment samples and corresponding organic extracts in order to identify the impact of different exposure pathways on sediment toxicity. MATERIALS AND METHODS: Danio rerio embryos were exposed to sublethal concentrations of three sediment samples from the Danube River, Germany. The sediment samples were investigated both as freeze-dried samples and as organic extracts. Silica dust and a process control of the extraction procedure were used as references. After exposure, mRNA was isolated and changes in profiles of gene expression levels were examined by an oligonucleotide microarray. The microarray results were compared with bioassays, chemical analysis of the sediments and profiles of gene expression levels induced by several single substances. RESULTS AND DISCUSSION: The microarray approach elucidated significant changes in the abundance of transcripts in exposed zebrafish embryos compared to the references. Generally, results could be related to Ah-receptor-mediated effects as confirmed by bioassays and chemical analysis of dioxin-like contaminants, as well as to exposure to stress-inducing compounds. Furthermore, the results indicated that mixtures of chemicals, as present in sediment and extract samples, result in complex changes of gene expression level profiles difficult to compare with profiles induced by single chemical substances. Specifically, patterns of transcript abundances were less influenced by the chemical composition at the sampling site compared t the method of exposure (sediment/extract. This effect might be related to different bioavailability of chemicals. CONCLUSIONS: The apparent

Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model

Directory of Open Access Journals (Sweden)

Hu YL

2011-12-01

Full Text Available Yu-Lan Hu1, Wang Qi1, Feng Han2, Jian-Zhong Shao3, Jian-Qing Gao11Institute of Pharmaceutics, College of Pharmaceutical Sciences, 2Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, 3College of Life Sciences, Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Zhejiang University, Hangzhou, Zhejiang, People's Republic of ChinaBackground: Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In the present study, the zebrafish model was used for a safety evaluation of this nanocarrier.Methods: Chitosan nanoparticles with two particle sizes were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles of different concentrations were incubated with zebrafish embryos, and ZnO nanoparticles were used as the positive control.Results: Embryo exposure to chitosan nanoparticles and ZnO nanoparticles resulted in a decreased hatching rate and increased mortality, which was concentration-dependent. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Furthermore, embryos exposed to chitosan nanoparticles showed an increased rate of cell death, high expression of reactive oxygen species, as well as overexpression of heat shock protein 70, indicating that chitosan nanoparticles can cause physiological stress in zebrafish. The results also suggest that the toxicity of biodegradable nanocarriers such as chitosan nanoparticles must be addressed, especially considering the in vivo distribution of these nanoscaled particles.Conclusion: Our results add new insights into the potential toxicity of nanoparticles produced by

Embryo-maternal communication

DEFF Research Database (Denmark)

Østrup, Esben; Hyttel, Poul; Østrup, Olga

2011-01-01

Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms dire...... directing the placentation. An increasing knowledge of the embryo-maternal communication might not only help to improve the fertility of our farm animals but also our understanding of human health and reproduction.......Communication during early pregnancy is essential for successful reproduction. In this review we address the beginning of the communication between mother and developing embryo; including morphological and transcriptional changes in the endometrium as well as epigenetic regulation mechanisms...

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

International Nuclear Information System (INIS)

Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

2014-01-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes

Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Malpass, Gloria E., E-mail: gloria.malpass@gmail.com [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu [Department of Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States); Prasad, G.L., E-mail: prasadg@rjrt.com [R and D Department, R.J. Reynolds Tobacco Company, Winston-Salem, NC 27102 (United States); Howlett, Allyn C., E-mail: ahowlett@wakehealth.edu [Department of Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157 (United States)

2014-09-01

Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

Survival and development of horseshoe crab (Limulus polyphemus) embryos and larvae in hypersaline conditions.

Science.gov (United States)

Ehlinger, Gretchen S; Tankersley, Richard A

2004-04-01

The horseshoe crab Limulus polyphemus spawns in the mid- to upper intertidal zone where females deposit eggs in nests below the sediment surface. Although adult crabs generally inhabit subtidal regions of estuaries with salinities from 5 to 34 ppt, developing embryos and larvae within nests are often exposed to more extreme conditions of salinity and temperature during summer spawning periods. To test whether these conditions have a negative impact on early development and survival, we determined development time, survival, and molt cycle duration for L. polyphemus embryos and larvae raised at 20 combinations of salinity (range: 30-60 ppt) and temperature (range: 25-40 degrees C). Additionally, the effect of hyperosmotic and hypoosmotic shock on the osmolarity of the perivitelline fluid of embryos was determined at salinities between 5 and 90 ppt. The embryos completed their development and molted at salinities below 60 ppt, yet failed to develop at temperatures of 35 degrees C or higher. Larval survival was high at salinities of 10-70 ppt but declined significantly at more extreme salinities (i.e., 5, 80, and 90 ppt). Perivitelline fluid remained nearly isoosmotic over the range of salinities tested. Results indicate that temperature and salinity influence the rate of crab development, but only the extremes of these conditions have an effect on survival.

Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos.

Science.gov (United States)

Rios, G L; Mucci, N C; Kaiser, G G; Alberio, R H

2010-03-01

The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, Pstraw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.

AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Massarsky, Andrey, E-mail: andrey.massarsky@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Bone, Audrey J. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Dong, Wu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); School of Animal Science and Technology, Inner Mongolia Provincial Key Laboratory for Toxicants and Animal Disease, Inner Mongolia University for the Nationalities, Tongliao, Inner Mongolia 028000 (China); Hinton, David E. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Prasad, G.L. [RAI Services Company, Winston-Salem, NC 27101 (United States); Di Giulio, Richard T. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

2016-10-15

The zebrafish embryo has been proposed as a ‘bridge model’ to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6 h post fertilization (hpf) until 96 hpf to TPM{sub 0.5} and TPM{sub 1.0} (corresponding to 0.5 and 1.0 μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96 hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity. - Highlights: • Total particulate matter (TPM) is the particulate phase of cigarette smoke. • Zebrafish is proposed as a ‘bridge model’ to study the effects of TPM. • We investigate the roles of antioxidant and aryl hydrocarbon receptor (AHR) pathways. �

Oral Morphine Consumption Reduces Lens Development in Rat Embryos

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Hossein Bahadoran

2012-07-01

Full Text Available Objective: Consumption of morphine, during pregnancy, in addition to inducing defects in the mother’s nervous system function, caused defects or delays in the formation and evolution of embryonic visual system. In the present study, changes in lens development was assessed in embryos exposed in utero to morphine. Material and Methods: Female Wistar rats (250-300 g were mated with male rats and pregnancy was determined by sperm observation in vaginal smear. This day was considered as embryonic day zero (E0. The females were then divided randomly into the experimental and the control groups. The control group received tap water and the experimental group received morphine (0.05 mg/ml in their water. On embryonic day 13 ( E13, blood samples were collected from the retro-orbital sinus of all animals for plasma corticosterone detection. On embryonic day 17(E17, the animals were killed by an overdose of chloroform and the embryos were taken out surgically. The embryos were fixed in 10% formalin for 30 days. At this time, the head of the embryos were removed for tissue processing and Hematoxylin- Eosin (H&E staining. The samples were evaluated using light microscope and MOTIC software. Results: Our data indicated that plasma corticosterone level was dramatically increased and the lens was thinner in the experimental group. (Although the proliferation of lens cells increased in the experiment group but that lens had delay in removing the proliferated and elongation cells with abnormal density in the lateral part of the lens in compare with control group. I have no idea what the authors are stating here. Moreover, the opening of the eyelids was delayed in the off springs of the mothers who received morphine. Conclusions: This study showed that morphine consumption during pregnancy leads to defects in fetal visual system development, particularly in the lens, and eyelids.

Direct evidence that radiation induced micronuclei of early embryos require a mitosis for expression

International Nuclear Information System (INIS)

Mueller, W.U.; Schlusen, I.; Streffer, C.

1991-01-01

The naturally synchronous development of early mouse embryos was exploited to address the question, whether micronuclei require a mitosis for expression or whether they can be expressed in the same cell cycle, in which exposure to X-rays or caffeine took place. Experiments with 2-cell and with 4-cell embryos showed that micronulcei are expressed only if a mitosis is completed. There was no indication, even after doses up to 20 Gy, that micronuclei can be expressed before the mitosis was reached, which followed exposure. Furthermore, no nuclear fragmentation pointing to apoptosis could be detected in the cycle, in which cells were exposed. The same results were obtained when caffeine (5 mM) was used as micronucleus inducing agent. (orig.)

Effect of periodontal dressings on human gingiva fibroblasts in vitro

International Nuclear Information System (INIS)

Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E.

1989-01-01

In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings

The effect of extremely low frequency electromagnetic fields on the chromosomal instability in bleomycin treated fibroblast cells

International Nuclear Information System (INIS)

Cho, Yoon Hee; Kim, Yang Jee; Lee, Joong Won; Kim, Gye Eun; Chung, Hai Won

2008-01-01

In order to determine the effect of Extremely Low Frequency ElectroMagnetic Fields (ELF-EMF) on the frequency of MicroNuclei (MN), aneuploidy and chromosomal rearrangement induced by BLeoMycin (BLM) in human fibroblast cells, a 60 Hz ELF-EMF of 0.8 mT field strength was applied either alone or with BLM throughout the culture period and a micronucleus-centromere assay was performed. Our results indicate that the frequencies of MN, aneuploidy and chromosomal rearrangement induced by BLM increased in a dose-dependent manner. The exposure of cells to 0.8 mT ELF-EMF followed by BLM exposure for 3 hours led to significant increases in the frequencies of MN and aneuploidy compared to BLM treatment for 3 hours alone (p<0.05), but no significant difference was observed between field exposed and sham exposed control cells. The obtained results suggest that low density ELF-EMF could act as enhancer of the initiation process of BLM rather than as an initiator of mutagenic effects in human fibroblast

Is mandating elective single embryo transfer ethically justifiable in young women?

Directory of Open Access Journals (Sweden)

Kelton Tremellen

2015-12-01

Full Text Available Compared with natural conception, IVF is an effective form of fertility treatment associated with higher rates of obstetric complications and poorer neonatal outcomes. While some increased risk is intrinsic to the infertile population requiring treatment, the practice of multiple embryo transfer contributes to these complications and outcomes, especially concerning its role in higher order pregnancies. As a result, several jurisdictions (e.g. Sweden, Belgium, Turkey, and Quebec have legally mandated elective single-embryo transfer (eSET for young women. We accept that in very high-risk scenarios (e.g. past history of preterm delivery and poor maternal health, double-embryo transfer (DET should be prohibited due to unacceptably high risks. However, we argue that mandating eSET for all young women can be considered an unacceptable breach of patient autonomy, especially since DET offers certain women financial and social advantages. We also show that mandated eSET is inconsistent with other practices (e.g. ovulation induction and intrauterine insemination–ovulation induction that can expose women and their offspring to risks associated with multiple pregnancies. While defending the option of DET for certain women, some recommendations are offered regarding IVF practice (e.g. preimplantation genetic screening and better support of IVF and maternity leave to incentivise patients to choose eSET.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

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Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

Science.gov (United States)

Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

Propagation of avian metapneumovirus subtypes A and B using chicken embryo related and other cell systems.

Science.gov (United States)

Coswig, Lia Treptow; dos Santos, Márcia Bianchi; Hafez, Hafez Mohamed; Ferreira, Helena Lage; Arns, Clarice Weis

2010-07-01

Primary isolation of avian metapneumovirus (aMPV) is carried out using tracheal organ culture (TOC) or chicken embryonated eggs with subsequent adaptation in chicken embryo fibroblasts (CEF) or Vero cultures. This study was conducted to evaluate six different cell lines and two avian culture systems for the propagation of aMPV subtypes A and B. The chicken embryo related (CER) cells were used successfully for primary isolation. In addition to Vero and baby hamster kidney (BHK-21) cells, CER cells were also shown to be the most appropriate for propagation of aMPV considering high titres. Propagation of A and B subtypes in CEF and TOC remained efficient after the primary isolation and several passages of viruses in the CER cell line. The growth curves were created using CER, Vero and BHK-21 cell lines. Compared with growth, both yielded higher titres in CER cells during the first 30 h after infection, but no significant difference was observed in the results obtained from CER and Vero cells. This data show that CER cells are adequate for aMPV subtypes A and B propagation, giving similar results to Vero cells. Copyright 2010 Elsevier B.V. All rights reserved.

Ten years investigation on radiological exposures to the embryo and fetus in pregnant women of Iran

International Nuclear Information System (INIS)

Arbabi, A.

2001-01-01

Over 1340 Iranian pregnant patients, exposed to diagnostic x-rays during 1984 to 1994, referred to me for investigation and estimation of the absorbed dose to their embryo or fetus. They were almost all the patients exposed to x-rays in the whole country in 10 years. Two sets of questionnaires were filed for each patient and all the exposing condition and setting information obtained from the radiology centers concerned. The absorbed dose to embryo or fetus accurately calculated and in some cases measured in a phantom for each patient. The youngest patient at the time of irradiation was 15 and the oldest was 51 with an average of 28.624±5.961 years old. The AP and lateral thickness of patient's abdomen on average were 18.078±0.162 cm and 24.630±8.365 cm respectively. The average weight of patients was 59.285±12.945 kg. ranging from 30 kg to 122 kg. The marriage age of the patients on average was 19.398±4.107 years ranging from 9 to 42 years old. The average age of fetus when exposed to x-rays was 31.22±18.76 days. About 20% of the patients had exposures in 2 to 4 more sessions. The average fetal dose was 0.68±0.381 cGy with over 37% from 1 to 9 cGy. (author)

Effect of embryo density on in vitro developmental characteristics of bovine preimplantative embryos with respect to micro and macroenvironments.

Science.gov (United States)

Hoelker, M; Rings, F; Lund, Q; Phatsara, C; Schellander, K; Tesfaye, D

2010-10-01

To overcome developmental problems as a consequence of single embryo culture, the Well of the Well (WOW) culture system has been developed. In this study, we aimed to examine the effect of embryo densities with respect to both microenvironment and macroenvironment on developmental rates and embryo quality to get a deeper insight into developmentally important mechanisms. WOW diameter and depth significantly affected developmental rates (p < 0.05). WOWs with diameter of 500 μm reached significantly higher blastocyst rates (32.5 vs 21.1% vs 20.3%) compared to embryos cultured in WOWs of 300 μm diameter or plain cultured controls. Embryos cultured in WOWs with 700 μm depth reached significant higher developmental rates compared with embryos cultured in WOWs of 300 μm depth and control embryos (30.6 vs 22.6% vs 20.3%). Correlation of the embryo per WOW volume with developmental rates was higher (r(2) = 0.92, p = 0.0004) than correlation of WOW diameter or WOW depth with developmental rates. However, the embryo per WOW volume did not affect differential cell counts. An embryo per culture dish volume of 1 : 30 μl was identified to be optimal when the embryo per WOW volume was 1 : 0.27 μl increasing developmental rates up to the level of mass embryo production. Giving the opportunity to track each embryo over the complete culture period while keeping high developmental rates with normal mitotic dynamics, the results of this work will provide benefit for the single culture of embryos in human assisted reproduction, mammalian embryos with high economic interest as well as for scientific purpose. © 2009 Blackwell Verlag GmbH.

Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

International Nuclear Information System (INIS)

An, Caiyan; Sato, Koichi; Wu, Taoya; Bao, Muqiri; Bao, Liang; Tobo, Masayuki; Damirin, Alatangaole

2015-01-01

The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

Extracellular acidification synergizes with PDGF to stimulate migration of mouse embryo fibroblasts through activation of p38MAPK with a PTX-sensitive manner

Energy Technology Data Exchange (ETDEWEB)

An, Caiyan [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China); Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Clinical Medicine Research Center of the Affiliated Hospital, Inner Mongolia Medical University, Hohhot, Inner Mongolia (China); Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Wu, Taoya; Bao, Muqiri; Bao, Liang [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China); Tobo, Masayuki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Damirin, Alatangaole, E-mail: bigaole@imu.edu.cn [Department of Biochemistry and Molecular Biology, College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia (China)

2015-05-01

The elucidation of the functional mechanisms of extracellular acidification stimulating intracellular signaling pathway is of great importance for developing new targets of treatment for solid tumors, and inflammatory disorders characterized by extracellular acidification. In the present study, we focus on the regulation of extracellular acidification on intracellular signaling pathways in mouse embryo fibroblasts (MEFs). We found extracellular acidification was at least partly involved in stimulating p38MAPK pathway through PTX-sensitive behavior to enhance cell migration in the presence or absence of platelet-derived growth factor (PDGF). Statistical analysis showed that the actions of extracellular acidic pH and PDGF on inducing enhancement of cell migration were not an additive effect. However, we also found extracellular acidic pH did inhibit the viability and proliferation of MEFs, suggesting that extracellular acidification stimulates cell migration probably through proton-sensing mechanisms within MEFs. Using OGR1-, GPR4-, and TDAG8-gene knock out technology, and real-time qPCR, we found known proton-sensing G protein-coupled receptors (GPCRs), transient receptor potential vanilloid subtype 1 (TRPV1), and acid-sensing ion channels (ASICs) were unlikely to be involved in the regulation of acidification on cell migration. In conclusion, our present study validates that extracellular acidification stimulates chemotactic migration of MEFs through activation of p38MAPK with a PTX-sensitive mechanism either by itself, or synergistically with PDGF, which was not regulated by the known proton-sensing GPCRs, TRPV1, or ASICs. Our results suggested that others proton-sensing GPCRs or ion channels might exist in MEFs, which mediates cell migration induced by extracellular acidification in the presence or absence of PDGF. - Highlights: • Acidic pH and PDGF synergize to stimulate MEFs migration via Gi/p38MAPK pathway. • Extracellular acidification inhibits the

Metabolite profiling of somatic embryos of Cyclamen persicum in comparison to zygotic embryos, endosperm and testa

Directory of Open Access Journals (Sweden)

Traud eWinkelmann

2015-08-01

Full Text Available Somatic embryogenesis has been shown to be an efficient in vitro plant regeneration system for many crops such as the important ornamental plant Cyclamen persicum, for which this regeneration pathway of somatic embryogenesis is of interest for the vegetative propagation of parental lines as well as elite plants. However, somatic embryogenesis is not commercially used in many crops due to several unsolved problems, such as malformations, asynchronous development, deficiencies in maturation and germination of somatic embryos. In contrast, zygotic embryos in seeds develop and germinate without abnormalities in most cases. Instead of time-consuming and labor-intensive experiments involving tests of different in vitro culture conditions and plant growth regulator supplements, we follow a more directed approach. Zygotic embryos served as a reference and were compared to somatic embryos in metabolomic analyses allowing the future optimization of the in vitro system. The aims of this study were to detect differences in the metabolite profiles of torpedo stage somatic and zygotic embryos of C. persicum. Moreover, major metabolites in endosperm and testa were identified and quantified.Two sets of extracts of two to four biological replicates each were analyzed. In total 52 metabolites were identified and quantified in the different tissues. One of the most significant differences between somatic and zygotic embryos was that the proline concentration in the zygotic embryos was about 40 times higher than that found in somatic embryos. Epicatechin, a scavenger for reactive oxygen species, was found in highest abundance in the testa. Sucrose, the most abundant metabolite was detected in significantly higher concentrations in zygotic embryos. Also, a yet unknown trisaccharide, was significantly enriched in zygotic embryos.

Increased viability of fibroblasts when pretreated with ceria nanoparticles during serum deprivation.

Science.gov (United States)

Genier, Francielli S; Bizanek, Maximilian; Webster, Thomas J; Roy, Amit K

2018-01-01

Conditions of cellular stress are often the cause of cell death or dysfunction. Sustained cell stress can lead to several health complications, such as extensive inflammatory responses, tumor growth, and necrosis. To prevent disease and protect human tissue during these conditions and to avoid medication side effects, nanomaterials with unique characteristics have been applied to biological systems. This paper introduces the pretreatment in human dermal fibroblasts with cerium oxide nanoparticles during nutritional stress. For this purpose, human dermal fibroblast cells received cell culture media with concentrations of 250 µg/mL and 500 µg/mL of nano-cerium oxide before being exposed to 24, 48, and 72 hours of serum starvation. Contrast images demonstrated higher cell confluence and cell integrity in cells pretreated with ceria nanoparticles compared to untreated cells. It was confirmed by MTS assay after 72 hours of serum starvation that higher cell viability was achieved with ceria nanoparticles. The results demonstrate the potential of cerium oxide nanoparticles as protective agents during cellular starvation.

In vitro blood and fibroblast responses to BisGMA-TEGDMA/bioactive glass composite implants.

Science.gov (United States)

Abdulmajeed, Aous A; Kokkari, Anne K; Käpylä, Jarmo; Massera, Jonathan; Hupa, Leena; Vallittu, Pekka K; Närhi, Timo O

2014-01-01

This in vitro study was designed to evaluate both blood and human gingival fibroblast responses to bisphenol A-glycidyl methacrylate-triethyleneglycol dimethacrylate (BisGMA-TEGDMA)/bioactive glass (BAG) composite, aimed to be used as composite implant abutment surface modifier. Three different types of substrates were investigated: (a) plain polymer (BisGMA 50 wt%-TEGDMA 50 wt%), (b) BAG-composite (50 wt% polymer + 50 wt% fraction of BAG-particles, <50 μm), and (c) plain BAG plates (100 wt% BAG). The blood response, including the blood-clotting ability and platelet adhesion morphology were evaluated. Human gingival fibroblasts were plated and cultured on the experimental substrates for up to 10 days, then the cell proliferation rate was assessed using AlamarBlue assay™. The BAG-composite and plain BAG substrates had a shorter clotting time than plain polymer substrates. Platelet activation and aggregation were most extensive, qualitatively, on BAG-composite. Analysis of the normalized cell proliferation rate on the different surfaces showed some variations throughout the experiment, however, by day 10 the BAG-composite substrate showed the highest (P < 0.001) cell proliferation rate. In conclusion, the presence of exposed BAG-particles enhances fibroblast and blood responses on composite surfaces in vitro.

Effect of the association of IGF-I, IGF-II, bFGF, TGF-beta1, GM-CSF, and LIF on the development of bovine embryos produced in vitro.

Science.gov (United States)

Neira, J A; Tainturier, D; Peña, M A; Martal, J

2010-03-15

This study examined the influence of the following growth factors and cytokines on early embryonic development: insulin-like growth factors I and II (IGF-I, IGF-II), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), granulocyte-macrophage colony-stimulating factor (GM-CSF), and leukemia inhibitory factor (LIF). Synthetic oviduct fluid (SOF) was used as the culture medium. We studied the development of bovine embryos produced in vitro and cultured until Day 9 after fertilization. TGF-beta1, bFGF, GM-CSF, and LIF used on their own significantly improved the yield of hatched blastocysts. IGF-I, bFGF, TGF-beta1, GM-CSF, and LIF significantly accelerated embryonic development, especially the change from the expanded blastocyst to hatched blastocyst stages. Use of a combination of these growth factors and cytokines (GF-CYK) in SOF medium produced higher percentages of blastocysts and hatched blastocysts than did use of SOF alone (45% and 22% vs. 24% and 12%; PGM-CSF, produces similar results to 10% fetal calf serum for the development of in vitro-produced bovine embryos. This entirely synthetic method of embryo culture has undeniable advantages for the biosecurity of embryo transfer. Copyright 2010 Elsevier Inc. All rights reserved.

Combined effect of radiation and polytriu insecticide on the development of rat embryo

Energy Technology Data Exchange (ETDEWEB)

Abu-Gabal, H A; Eid, F A; Gaber, S H [National Centre for Radiation Research Technology, Faculty of Science for Girls, Al-Azhar University. Cairo (Egypt)

1987-12-31

In the present study 9 groups of pregnant female rats were used. The 1st group served as the control. The 2nd and 3rd groups were orally administered polytrin insecticide:1/100 L D 50 on days 1 and 7 of gestation. Groups 4,5 and 6 were exposed to 2 Gy -rays on day 6, day 12 or days 6 and 12 of gestation. Groups 7,8 and 9 were exposed to 2 Gy gamma -ray on day 6, day 12 or days 6 and 12 during polytrin treatment. The results showed serious abnormal developmental changes as intrauterine death, gross malformations and relation when the embryos or fetuses were maternally exposed to -rays and/or treated with the pyrethroid compound. Moreover the malformations were higher and more drastic in the groups exposed to irradiation during polytrin treatment. These results are very important from the standpoint environmental pollution and radiation protection and safety. figs 23., 8 tabs.

Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

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Zhao Roong

2001-12-01

Full Text Available Abstract Background Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. Results Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. Conclusion Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This

Establishment of pregnancies with handmade cloning porcine embryos reconstructed with fibroblasts containing an Alzheimer's disease gene

DEFF Research Database (Denmark)

Kragh, P; Li, J; Du, Y

2008-01-01

Somatic cell nuclear transfer (SCNT) offers the possibility of pig transgenesis. Importantly, specific genetic manipulations can be performed in donor cells before SCNT to derive pig models for specific human genetic diseases, including the neurodegenerative disorder Alzheimer's disease (AD......). In the present study, we established pregnancies after transfer of SCNT blastocysts produced by the handmade cloning (HMC) technique. The blastocysts were transgenic for a human gene, amyloid precursor protein gene with the 'Swedish mutation' (APPsw), causing AD. For transgenesis, minipig fibroblasts were...... ovaries of slaughtered sows and matured for 41 h. Subsequently, the cumulus cells were removed in hyaluronidase, and zonae pellucidae were partially digested by incubation in pronase. Oocytes with a visible polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm adjacent...

Feel, smell and see in an egg: emergence of perception and learning in an immature invertebrate, the cuttlefish embryo.

Science.gov (United States)

Romagny, Sébastien; Darmaillacq, Anne-Sophie; Guibé, Mathieu; Bellanger, Cécile; Dickel, Ludovic

2012-12-01

It is now well established that prenatal sensory experience affects development itself and has long-term consequences in terms of postnatal behavior. This study focused on the functionality of the sensory system in cuttlefish in ovo. Embryos of stage 23, 25 and 30 received a tactile, chemical or visual stimulus. An increase of mantle contraction rhythm was taken to indicate a behavioral response to the stimulus. We clearly demonstrated that tactile and chemical systems are functional from stage 23, whereas the visual system is functional only from stage 25. At stage 25 and 30, embryos were also exposed to a repeated light stimulus. Stage 30 embryos were capable of habituation, showing a progressive decrease in contractions across stimulations. This process was not due to fatigue as we observed response recovery after a dishabituation tactile stimulus. This study is the first to show that cuttlefish embryos behaviorally respond to stimuli of different modalities and that the visual system is the last to become functional during embryonic development, as in vertebrate embryos. It also provides new evidence that the memory system develops in ovo in cuttlefish.

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

International Nuclear Information System (INIS)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

2011-01-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

Energy Technology Data Exchange (ETDEWEB)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

2011-07-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Cytotoxicity of Betel leaf (Piper betel L. against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells

Directory of Open Access Journals (Sweden)

Suprapto Ma’at

2012-06-01

Full Text Available Background: Betel leaf (Piper betel L. has been used in modern and traditional medicine as antiseptic, antibacterial, and also prevention of plaque accumulation, but it still can stimulate cancer in lime-piper betel quid. Betel leaf also has anti-inflammatory properties. Purpose: The purpose of this study was examine the cytotoxicity of Betel leaf extract (BLE against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMC stimulated with LPS. Methods: MTT assay was used to investigate the survival rate of the culture with the survival rate result of the given culture extract 4%, 2% and 1% about 82%, 83.4% and 85%. There was no significant difference between treatment with various concentrations of the extract and the control (p>0.05. To evaluate the effect of Betel leaf extracts on the production of cytokines, proinflammatory was conducted by incubating the extracts of betel leaf with peripheral blood mononuclear cells stimulated with lipopolysaccharide. Peripheral blood mononuclear cells were obtained from healthy volunteers isolated by density centrifugation method using Ficoll-Hypaque. Once coupled with various concentrations of betel leaf extract and lipopolysaccharide, and then incubated for 24 hours, the culture supernatant was used to determine the level of IFN-γ and TNF-α by ELISA method. Results: It is known that the survival rates of BLE 4%, 2% and 1% were 82%, 83.4% and 85%. There was no significant of difference between several concentrations of BLE and those in the control group (p>0.05. The production of IFN-γ and TNF-α stimulated with LPS was no significant difference between BLE 4%, 2% and 1% and that in the control group (p>0.05. Conclusion: It can be concluded that BLE is not toxic against primary culture of chicken embryo fibroblast, and the production of IFN-γ and TNF-α by PBMC was not affected by BLE.Latar belakang: Daun

Effect of Radiofrequency Radiation Emitted from 2G and 3G Cell Phone on Developing Liver of Chick Embryo – A Comparative Study

Science.gov (United States)

Swer, Rijied Thompson; Anbalagan, J.; Rajesh, Bhargavan

2017-01-01

Introduction The increasing scientific evidence of various health hazards on exposure of Radiofrequency Radiation (RFR) emitted from both the cell phones and base stations have caused significant media attention and public discussion in recent years. The mechanism of interaction of RF fields with developing tissues of children and fetuses may be different from that of adults due to their smaller physical size and variation in tissue electromagnetic properties. The present study may provide an insight into the basic mechanisms by which RF fields interact with developing tissues in an embryo. Aim To evaluate the possible tissue and DNA damage in developing liver of chick embryo following chronic exposure to Ultra-High Frequency/Radiofrequency Radiation (UHF/RFR) emitted from 2G and 3G cell phone. Materials and Methods Fertilized chick embryos were incubated in four groups. Group A-experimental group exposed to 2G radiation (60 eggs), Group B- experimental group exposed to 3G radiation (60 eggs), Group C- sham exposed control group (60 eggs) and Group D– control group (48 eggs). On completion of scheduled duration, the embryos were collected and processed for routine histological studies to check structural changes in liver. The nuclear diameter and karyorrhexis changes of hepatocytes were analysed using oculometer and square reticule respectively. The liver procured from one batch of eggs from all the four groups was subjected to alkaline comet assay technique to assess DNA damage. The results were compared using one-way ANOVA test. Results In our study, the exposure of developing chick embryos to 2G and 3G cell phone radiations caused structural changes in liver in the form of dilated sinusoidal spaces with haemorrhage, increased vacuolations in cytoplasm, increased nuclear diameter and karyorrhexis and significantly increased DNA damage. Conclusion The chronic exposure of chick embryo liver to RFR emitted from 2G and 3G cell phone resulted in various structural

Nano-nutrition of chicken embryos

DEFF Research Database (Denmark)

Sawosz, Filip; Pineda, Lane Manalili; Hotowy, Anna

2013-01-01

It has been suggested that the quantity and quality of nutrients stored in the egg might not be optimal for the fast rate of chicken embryo development in modern broilers, and embryos could be supplemented with nutrients by in ovo injection. Recent experiments showed that in ovo feeding reduces...... broiler eggs was randomly divided into a Control group without injection and injected groups with hydrocolloids of Nano-Ag, ATP or a complex of Nano-Ag and ATP (Nano-Ag/ATP). The embryos were evaluated on day 20 of incubation. The results indicate that the application of ATP to chicken embryos increases...

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

International Nuclear Information System (INIS)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

2005-01-01

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

In vitro production of buffalo embryos from stepwise vitrified immature oocytes.

Science.gov (United States)

Abd-Allah, Saber Mohammed

2009-01-01

This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs) were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 microl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7%) with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%), cleavage (47.6%) and buffalo embryo development (15.4%) produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05).

In vitro production of buffalo embryos from stepwise vitrified immature oocytes

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Saber Mohammed Abd-Allah

2009-09-01

Full Text Available This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7% with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%, cleavage (47.6% and buffalo embryo development (15.4% produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05.

Radionuclide transfer from mother to embryo

International Nuclear Information System (INIS)

Toader, M.; Vasilache, R.A.; Scridon, R.; Toader, M.L.

1998-01-01

The transfer of radionuclides from mother to embryo is still a matter of high interest. Therefore, the relation was investigated between the amount of radionuclides in the embryo and the dietary intake of the mother, this for two scenarios: a recurrent intake of variable amounts of radionuclides, and a long-term intake of a relatively constant amount of radionuclides, the radionuclide being 137 Cs. In the first case, the amount of radionuclides present in the embryo increases with the age of the embryo and with the intake of the mother. In the second case, no correlation could be found between the age of the embryo and its radioactive content; only the correlation between the intake of the mother and the radionuclide content of the embryo remained. (A.K.)

Manipulating early pig embryos.

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Niemann, H; Reichelt, B

1993-01-01

On the basis of established surgical procedures for embryo recovery and transfer, the early pig embryo can be subjected to various manipulations aimed at a long-term preservation of genetic material, the generation of identical multiplets, the early determination of sex or the alteration of the genetic make-up. Most of these procedures are still at an experimental stage and despite recent considerable progress are far from practical application. Normal piglets have been obtained after cryopreservation of pig blastocysts hatched in vitro, whereas all attempts to freeze embryos with intact zona pellucida have been unsuccessful. Pig embryos at the morula and blastocyst stage can be bisected microsurgically and the resulting demi-embryos possess a high developmental potential in vitro, whereas their development in vivo is impaired. Pregnancy rates are similar (80%) but litter size is reduced compared with intact embryos and twinning rate is approximately 2%. Pig blastomeres isolated from embryos up to the 16-cell stage can be grown in culture and result in normal blastocysts. Normal piglets have been born upon transfer of blastocysts derived from isolated eight-cell blastomeres, clearly underlining the totipotency of this developmental stage. Upon nuclear transfer the developmental capacity of reconstituted pig embryos is low and culture. Sex determination can be achieved either by separation of X and Y chromosome bearing spermatozoa by flow cytometry or by analysing the expression of the HY antigen in pig embryos from the eight-cell to morula stage. Microinjection of foreign DNA has been successfully used to alter growth and development of transgenic pigs, and to produce foreign proteins in the mammary gland or in the bloodstream, indicating that pigs can be used as donors for valuable human pharmaceutical proteins. Another promising area of gene transfer is the increase of disease resistance in transgenic lines of pigs. Approximately 30% of pig spermatozoa bind

Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

International Nuclear Information System (INIS)

Giometti, C.S.; Willard, K.E.; Anderson, N.L.

1982-01-01

Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with 125 I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture

Accurate and noninvasive embryos screening during in vitro fertilization (IVF) assisted by Raman analysis of embryos culture medium Accurate and noninvasive embryos screening during IVF

Science.gov (United States)

Shen, A. G.; Peng, J.; Zhao, Q. H.; Su, L.; Wang, X. H.; Hu, J. M.; Yang, J.

2012-04-01

In combination with morphological evaluation tests, we employ Raman spectroscopy to select higher potential reproductive embryos during in vitro fertilization (IVF) based on chemical composition of embryos culture medium. In this study, 57 Raman spectra are acquired from both higher and lower quality embryos culture medium (ECM) from 10 patients which have been preliminarily confirmed by clinical assay. Data are fit by using a linear combination model of least squares method in which 12 basis spectra represent the chemical features of ECM. The final fitting coefficients provide insight into the chemical compositions of culture medium samples and are subsequently used as criterion to evaluate the quality of embryos. The relative fitting coefficients ratios of sodium pyruvate/albumin and phenylalanine/albumin seem act as key roles in the embryo screening, attaining 85.7% accuracy in comparison with clinical pregnancy. The good results demonstrate that Raman spectroscopy therefore is an important candidate for an accurate and noninvasive screening of higher quality embryos, which potentially decrease the time-consuming clinical trials during IVF.

The effects of green tea extract on teratogenicity induced by low frequency electromagnetic field on bone marrow Balb/C mice embryo

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Baharara Javad

2014-01-01

Full Text Available Introduction: Electromagnetic fields produce free radicals which might be teratogen. Camellia sinensis is rich in natural antioxidants and antioxidants can neutralize free radicals effects. In present research the effect of C. sinensis extract in reduction of teratogenicity induced by electromagnetic field with 50 gauss intensity was studied on bone marrow of Balb/C mice fetuses. Methods: In this experimental study, 24 Balb/C pregnant mice were randomly divided into four groups: control, sham exposed (off position, experimental 1 (electromagnetic field with 50-gauss intensity and experimental 2 (treatment by C. sinensis extract + electromagnetic field with 50-gauss intensity. After treatment period, the bone marrow aspirates of Balb/C mice embryos were prepared and studied by Giemsa. The quantitative data were analyzed by Kruskal-Wallis and Kolmogorov- Smirnov using SPSS16 software at the level of p<0.05. Results: The mean number of promyelocytes, myelocytes, erythrocytes, necrotic and apoptotic cells in experimental group1 compared with sham exposed embryos showed significant increase but the mean number of eosinophils in experimental group 1 compared with sham exposed embryos showed significant decrease. The mean number of promyelocyte and erythrocyte in experimental group 2 compared with experimental group 1 showed significant decrease. The mean of necrotic and apoptotic cells, in experimental group 2 compared with experimental group 1 showed significant increase. Conclusion: Usage of C. sinensis can decrease the damage due to teratogenicity induced by low frequency electromagnetic field in some cells.

The effect of oil sands tailings pond sediments on embryo-larval walleye (Sander vitreus).

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Raine, J C; Turcotte, D; Tumber, V; Peru, K M; Wang, Z; Yang, C; Headley, J V; Parrott, J L

2017-10-01

Walleye (Sander vitreus) are a commercially important North American fish species that inhabit the Athabasca River. This river flows through the Athabasca oil sands where natural sources of bitumen erode from the McMurray formation. Little information is available on responses of walleye embryos to oil sands tailings pond sediments in a laboratory setting. The current study describes the design and implementation of a daily-renewal bioassay to assess the potential effects of tailings pond sediments from the Athabasca oil sands area on walleye development. Developing walleye embryos were exposed to increasing concentrations of two tailings pond sediments (collected in the Athabasca oil sands area) until the completion of yolk absorption in control fish. Sediments from the tailings pond represent a mixture of polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs. During the 31 day exposure, the walleye were examined for mortalities, weight, length and developmental abnormalities to provide an initial evaluation of the effects of the oil sands tailings pond sediments. Walleye embryo survival differed between the tailings pond sediments, and survival decreased with increasing sediment concentration. Alkylated PAH content differed between the two tailings pond sediments and lower embryo survival corresponded to higher total and alkylated PAH content. Tailings pond sediment-exposed walleye exhibited a delay in development, as well as increased percentages of larvae with heart and yolk sac edema, and cranial and spinal malformations. These abnormalities in development are often associated with PAH and alkylated PAH exposure. This study provides an exposure design that can be used to assess sediment toxicity to early developmental stages of a fish species not commonly tested in the lab, and lays the groundwork for future studies with this and other difficult-to-culture species. These results offer information on the potential effects of tailings pond sediments

What Drives Embryo Development? Chromosomal Normality or Mitochondria?

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A. Bayram

2017-01-01

Full Text Available Objective. To report the arrest of euploid embryos with high mtDNA content. Design. A report of 2 cases. Setting. Private fertility clinic. Patients. 2 patients, 45 and 40 years old undergoing IVF treatment. Interventions. Mature oocytes were collected and vitrified from two ovarian stimulations. Postthaw, survived mature oocytes underwent fertilization by intracytoplasmic sperm injection (ICSI. Preimplantation genetic screening (PGS and mitochondrial DNA (mtDNA copy number were done using next generation sequencing (NGS. The only normal embryo among the all-biopsied embryos had the highest “Mitoscore” value and was the only arrested embryo in both cases. Therefore, the embryo transfer was cancelled. Main Outcome Measures. Postthaw survival and fertilization rate, embryo euploidy, mtDNA copy number, and embryo development. Results. In both patients, after PGS only 1 embryo was euploid. Both embryos had the highest mtDNA copy number from all tested embryos and both embryos were arrested on further development. Conclusions. These cases clearly demonstrate the lack of correlation between mtDNA value (Mitoscore and chromosomal status of embryo.

A novel role of EMMPRIN/CD147 in transformation of quiescent fibroblasts to cancer-associated fibroblasts by breast cancer cells

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Xu, Jing; Lu, Yang; Qiu, Songbo; Chen, Zhi-Nan; Fan, Zhen

2013-01-01

We tested the novel hypothesis that EMMPRIN/CD147, a transmembrane glycoprotein overexpressed in breast cancer cells, has a previously unknown role in transforming fibroblasts to cancer-associated fibroblasts, and that cancer-associated fibroblasts in turn induce epithelial-to-mesenchymal transition of breast cancer cells. Co-culture of fibroblasts with breast cancer cells or treatment of fibroblasts with breast cancer cell conditioned culture medium or recombinant EMMPRIN/CD147 induced expression of α-SMA in the fibroblasts in an EMMPRIN/CD147-dependent manner and promoted epithelial-to-mesenchymal transition of breast cancer cells and enhanced cell migration potential. These findings support a novel role of EMMPRIN/CD147 in regulating the interaction between cancer and stroma. PMID:23474495

mTOR complex 2 phosphorylates IMP1 cotranslationally to promote IGF2 production and the proliferation of mouse embryonic fibroblasts

DEFF Research Database (Denmark)

Dai, Ning; Christiansen, Jan; Nielsen, Finn

2013-01-01

uncover a new mechanism by which mTOR regulates organismal growth by promoting IGF2 production in the mouse embryo through mTORC2-catalyzed cotranslational IMP1/IMP3 phosphorylation. Inasmuch as TORC2 is activated by association with ribosomes, the present results indicate that mTORC2-catalyzed...... production, and diminished proliferation. The proliferation of the IMP1-null fibroblasts can be restored to wild-type levels by IGF2 in vitro or by re-expression of IMP1, which corrects the defects in IGF2 RNA splicing and translation. The ability of IMP1 to correct these defects is dependent on IMP1...

Theory about the Embryo Cryo-Treatment.

Science.gov (United States)

Vladimirov, Iavor K; Tacheva, Desislava; Diez, Antonio

2017-04-01

To create hypothesis, which can give a logical explanation related to the benefits of freezing/thawing embryos. Cryopreservation is not only a technology used for storing embryos, but also a method of embryo treatment that can potentially improve the success rate in infertile couples. From the analysis of multiple results in assisted reproductive technology, which have no satisfactory explanation to date, we found evidence to support a 'therapeutic' effect of the freezing/thawing of embryos on the process of recovery of the embryo and its subsequent implantation. Freezing/thawing is a way to activate the endogenous survival and repair responses in preimplantation embryos. Several molecular mechanisms can explain the higher success rate of ET using thawed embryos compared to fresh ET in women of advanced reproductive age, the higher miscarriage rate in cases of thawed blastocyst ET compared to thawed ET at early cleavage embryo, and the higher perinatal parameters of born children after thawed ET. Embryo thawing induces a stress. Controlled stress is not necessarily detrimental, because it generates a phenomenon that is counteracted by several known biological responses aimed to repair mitochondrial damage of membrane and protein misfolding. The term for favorable biological responses to low exposures to stress is called hormesis. This thesis will summarize the role of cryopreservation in the activation of a hormetic response, preserving the mitochondrial function, improving survival, and having an impact on the process of implantation, miscarriage, and the development of pregnancy.

Dose-response relationship of cadmium or radiation-induced embryotoxicity in mouse whole embryo culture

International Nuclear Information System (INIS)

Nakashima, Kiyohito; Kawamata, Akitoshi; Matsuoka, Masato; Wakisaka, Takashi; Fujiki, Yoshishige

1988-01-01

Mouse embryos of B6C3F 1 strain were exposed in vitro to 1.2 to 2.2 μM cadmium chloride (Cd) or to 100 to 320 R x-rays, and the effects of the exposure on development were examined after 39 h of culture. Development of embryos was assessed from lethality, formation of the neural tube defect, diameter and protein of yolk sac, crown-rump and head lengths, embryonic protein, and number of somites. Incidence of the neural tube defect increased from 3.4 to 100% by 1.2 to 2.0 μM Cd, while embryo deaths increased from 13.8 to 93.3% by 2.0 to 2.2 μM Cd. Embryonic protein was significantly reduced at the teratogenic range, but the number of somites was only affected by 1.6 to 2.0 μM Cd. X-irradiation at 100 to 320 R induced the neural tube defect in 2.9 to 72.7% of the embryos. An embryolethal effect was observed only at the 320 R dose. Crown-rump and head lengths and embryonic protein were significantly affected at the teratogenic range, but the diameter and protein of yolk sac and number of somites were hardly affected. Cadmium- or radiation-induced response data of both teratogenicity and endpoints indicating inhibition of embryonic development were acceptably fitted to a linear log-probit regression. These regressions suggest that as an estimation of interference in development of embryos, embryonic protein and head length are sensitive endpoints while the number of somites is an insensitive criterion. (author)

Tributyltin induces premature hatching and reduces locomotor activity in zebrafish (Danio rerio) embryos/larvae at environmentally relevant levels.

Science.gov (United States)

Liang, Xuefang; Souders, Christopher L; Zhang, Jiliang; Martyniuk, Christopher J

2017-12-01

Tributyltin (TBT) is an organotin compound that is the active ingredient of many biocides and antifouling agents. In addition to its well established role as an endocrine disruptor, TBT is also associated with adverse effects on the nervous system and behavior. In this study, zebrafish (Danio rerio) embryos were exposed to environmentally relevant concentrations of TBT (0.01, 0.1, 1 nM) to determine how low levels affected development and behavior. Fish exposed to 1 nM TBT hatched earlier when compared to controls. Following a 96-h exposure, total swimming distance, velocity, and activity of zebrafish larvae were reduced compared to controls. To identify putative mechanisms for these altered endpoints, we assessed embryo bioenergetics and gene expression. We reasoned that the accelerated hatch time could be related to ATP production and energy, thus embryos were exposed to TBT for 24 and 48-h exposure prior to hatch. There were no differences among groups for endpoints related to bioenergetics (i.e. basal, ATP-dependent, and maximal respiration). To address mechanisms related to changes in behavioral activity, we measured transcripts associated with muscle function (myf6, myoD, and myoG) and dopamine signaling (th, dat, dopamine receptors) as dopamine regulates behavior. No transcript was altered in expression by TBT in larvae, suggesting that other mechanisms exist that may explain changes in higher level endpoints. These results suggest that endpoints related to the whole animal (i.e. timing of hatch and locomotor behavior) are more sensitive to environmentally-relevant concentrations of TBT compared to the molecular and metabolic endpoints examined here. Copyright © 2017 Elsevier Ltd. All rights reserved.

[The destiny of cryopreserved embryos].

Science.gov (United States)

Karpel, L; Achour-Frydman, N; Frydman, R; Flis-Trèves, M

2007-12-01

To know the psychological motivations of couples who keep their embryos so long (five years and more) and do not make a decision about them. We studied 84 couples refrained from making a decision on their cryopreserved embryos for at least five years. They were invited to fill out a questionnaire focusing on three points: the reasons of the indecision, their own representation of the cryopreserved embryos and their choice for the future: donation to another couple, to research, pregnancy or no solution for the moment. Mean (S.D.) women's and men's age were respectively, 38.8 (2.5)- and 41.3 (2.5)-years old. On average, three (1-9) embryos are preserved since 7.5 (5-12) years. Most of couples are parents. Four major reasons explain their attitudes: feeling of being too aged (25%), fear of a multiple pregnancy (45%), disagreement between members of couple (20%) and fear of failure (42.5%). Multiple choices were given to the future of the embryos: 25% wanted a pregnancy, 8% wanted to give them to infertile couples, 20% to research and 27.5% did not find any solution. Twenty percent were hesitating. The representation of those embryos is more symbolic than material. Most of the time, they see them like a potential child, a hope for the future or a brother or sister of their alive children. Those embryos are symbolized. They are a proof of fertility, a hope for another child. So, whatever the legal statement, couples will be in a dilemma because it is never easy for an infertile person to renounce to embryos, and the hope for children.

Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

International Nuclear Information System (INIS)

Story, M.T.

1989-01-01

To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue

Laboratory techniques for human embryos.

Science.gov (United States)

Geber, Selmo; Sales, Liana; Sampaio, Marcos A C

2002-01-01

This review is concerned with laboratory techniques needed for assisted conception, particularly the handling of gametes and embryos. Such methods are being increasingly refined. Successive stages of fertilization and embryogenesis require especial care, and often involve the use of micromanipulative methods for intracytoplasmic sperm injection (ICSI) or preimplantation genetic diagnosis. Embryologists must take responsibility for gamete collection and preparation, and for deciding on the means of insemination or ICSI. Embryos must be assessed in culture, during the 1-cell, cleaving and morula/blastocyst stages, and classified according to quality. Co-culture methods may be necessary. The best embryos for transfer must be selected and loaded into the transfer catheter. Embryos not transferred must be cryopreserved, which demands the correct application of current methods of media preparation, seeding and the correct speed for cooling and warming. Before too long, methods of detecting abnormal embryos and avoiding their transfer may become widespread.

In vitro embryo culture of rarely endangered musella lasiocarpa (musaceae) with embryo dormancy

International Nuclear Information System (INIS)

Anjun, T.

2014-01-01

Musella lasiocarpa (Musaceae) is an ornamental annually producing many viable seeds, but seldom recruited by seeds in the wild. One mature Musella seed has a small mushroom-shaped embryo without discernible organ differentiation. Therefore, freshly-harvested mature seeds are dormant. When the seeds gradually finished differentiation during warm stratification at 23 degree C, they germinated to 82%. Besides, extracted embryos from fresh seeds did not germinate on the basal medium of Murshige and Skoog medium (MS) supplemented with 3% sucrose and 0.8% agar, but they were induced to form calli and root by media. The optimum medium for inducing calli was MS + 1.0 mg/L 6-BA + 0.05 mg/L NAA + 100 mg/L Vc with the highest proliferation coefficient (7.3) in 35 days. Moreover, the embryos from the 6-month warm stratified seeds could proliferate on the suitable medium. The optimal medium for rooting was MS + 0.5 mg/L 2, 4-D + Vitamin C 100 mg/L. The results confirmed that both the embryo developmental stage and appropriate combination of chemicals significantly affected seed germination and In vitro embryo culture of this species. (author)

Effects of embryo-derived exosomes on the development of bovine cloned embryos.

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Pengxiang Qu

Full Text Available The developmental competence of in vitro cultured (IVC embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE, as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development, but also following growth to term (in vivo development. Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

Feminists on the inalienability of human embryos.

Science.gov (United States)

McLeod, Carolyn; Baylis, Francoise

2006-01-01

The feminist literature against the commodification of embryos in human embryo research includes an argument to the effect that embryos are "intimately connected" to persons, or morally inalienable from them. We explore why embryos might be inalienable to persons and why feminists might find this view appealing. But, ultimately, as feminists, we reject this view because it is inconsistent with full respect for women's reproductive autonomy and with a feminist conception of persons as relational, embodied beings. Overall, feminists should avoid claims about embryos' being inalienable to persons in arguments for or against the commodification of human embryos.

Molecular pathways in the bystander response of cells exposed to very low fluences of alpha particles

International Nuclear Information System (INIS)

Little, J.B.

2000-01-01

Full text: We have examined biological effects in cell populations exposed to very low mean doses of alpha radiation by which only a small fraction of the cells are actually traversed by an alpha particle. We showed earlier that an enhanced frequency of sister chromatid exchanges and HPRT mutations occur in the non-irradiated, 'bystander' cells. The frequency of mutations induced by a single alpha particle traversing the nucleus of a cell was increased nearly fivefold at the lowest fluence studied, a result of mutations occurring in bystander cells. This was associated with a similar increase in the induction of micronuclei, indicating the induction of DNA damage in bystander cells. In order to gain information concerning molecular pathways, we studied changes in gene expression in bystander cells in confluent cultures of human diploid fibroblasts or mouse embryo-derived fibroblasts (MEFs) by western analysis and in-situ immunofluorescence. The expression levels of p53, p21 Waf1 and p34 cdc2 were significantly modulated in bystander cells. The upregulation of p53 and p21 Waf1 did not occur in cultures irradiated at low density, and was markedly reduced in the presence of the gap junction inhibitor lindane. The importance of gap-junction mediated intercellular communication was confirmed in connexin-43 knockout MEFs. Western blot analyses and electrophoretic mobility shift assays indicate that the bystander response is suppressed by incubation with superoxide dismutase as well as an inhibitor of NADPH oxidase, and is associated with the induction of NFKB, suggesting the effect is mediated by oxidative stress. The stress-activated protein kinase p38 and its downstream effector ATF2 are also induced in bystander cells independent of oxidative stress. These results will be discussed in terms of whether activation of the p53 damage response pathway is the direct result of signaling from irradiated cells, or rather is a consequence of DNA induced damage in the bystander

Increased viability of fibroblasts when pretreated with ceria nanoparticles during serum deprivation

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Genier FS

2018-02-01

Full Text Available Francielli S Genier,1 Maximilian Bizanek,1 Thomas J Webster,1,2 Amit K Roy1,2 1Department of Chemical Engineering, Northeastern University, Boston, MA, USA; 2Wenzhou Institute of Biomaterials and Engineering (WIBE, Wenzhou University, Wenzhou, People’s Republic of China Abstract: Conditions of cellular stress are often the cause of cell death or dysfunction. Sustained cell stress can lead to several health complications, such as extensive inflammatory responses, tumor growth, and necrosis. To prevent disease and protect human tissue during these conditions and to avoid medication side effects, nanomaterials with unique characteristics have been applied to biological systems. This paper introduces the pretreatment in human dermal fibroblasts with cerium oxide nanoparticles during nutritional stress. For this purpose, human dermal fibroblast cells received cell culture media with concentrations of 250 µg/mL and 500 µg/mL of nano-cerium oxide before being exposed to 24, 48, and 72 hours of serum starvation. Contrast images demonstrated higher cell confluence and cell integrity in cells pretreated with ceria nanoparticles compared to untreated cells. It was confirmed by MTS assay after 72 hours of serum starvation that higher cell viability was achieved with ceria nanoparticles. The results demonstrate the potential of cerium oxide nanoparticles as protective agents during cellular starvation. Keywords: cerium oxide, nanoparticles, serum starvation, human dermal fibroblasts

Effect of 660 nm Light-Emitting Diode on the Wound Healing in Fibroblast-Like Cell Lines

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Myung-Sun Kim

2015-01-01

Full Text Available Light in the red to near-infrared (NIR range (630–1000 nm, which is generated using low energy laser or light-emitting diode (LED arrays, was reported to have a range of beneficial biological effects in many injury models. NIR via a LED is a well-accepted therapeutic tool for the treatment of infected, ischemic, and hypoxic wounds as well as other soft tissue injuries in humans and animals. This study examined the effects of exposure to 660 nm red LED light at intensities of 2.5, 5.5, and 8.5 mW/cm2 for 5, 10, and 20 min on wound healing and proliferation in fibroblast-like cells, such as L929 mouse fibroblasts and human gingival fibroblasts (HGF-1. A photo illumination-cell culture system was designed to evaluate the cell proliferation and wound healing of fibroblast-like cells exposed to 600 nm LED light. The cell proliferation was evaluated by MTT assay, and a scratched wound assay was performed to assess the rate of migrating cells and the healing effect. Exposure to the 660 nm red LED resulted in an increase in cell proliferation and migration compared to the control, indicating its potential use as a phototherapeutic agent.

An economic assessment of embryo diagnostics (Dx) - the costs of introducing non-invasive embryo diagnostics into IVF standard treatment practices.

Science.gov (United States)

Fugel, Hans-Joerg; Connolly, Mark; Nuijten, Mark

2014-10-09

New techniques in assessing oocytes and embryo quality are currently explored to improve pregnancy and delivery rates per embryo transfer. While a better understanding of embryo quality could help optimize the existing "in vitro fertilization" (IVF) therapy schemes, it is essential to address the economic viability of such technologies in the healthcare setting. An Embryo-Dx economic model was constructed to assess the cost-effectiveness of 3 different IVF strategies from a payer's perspective; it compares Embryo-Dx with single embryo transfer (SET) to elective single embryo transfer (eSET) and to double embryo transfer (DET) treatment practices. The introduction of a new non-invasive embryo technology (Embryo-Dx) associated with a cost up to €460 is cost-effective compared to eSET and DET based on the cost per live birth. The model assumed that Embryo-Dx will improve ongoing pregnancy rate/realize an absolute improvement in live births of 9% in this case. This study shows that improved embryo diagnosis combined with SET may have the potential to reduce the cost per live birth per couple treated in IVF treatment practices. The results of this study are likely more sensitive to changes in the ongoing pregnancy rate and consequently the live birth rate than the diagnosis costs. The introduction of a validated Embryo-Dx technology will further support a move towards increased eSET procedures in IVF clinical practice and vice versa.

EVALUATION OF ETHINYLESTRADIOL (EE2 EFFECT ON EMBRYO DEVELOPMENT IN COMMON CARP (CYPRINUS CARPIO

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GABI DUMITRESCU

2009-10-01

Full Text Available Worldwide, the scientific researches performed during the last years are focused on the determination of the negative effects caused by natural and antropogeneous chemical compounds on aquatic species; these species are more exposed to most pollutants than the land species, for the simple reason that the aquatic environment is the last destination for most residues. Our research team proposed to test the toxic effect caused by ethinylestradiol on embryo development in common carp (Cyprinus carpio. Common carp embryos were purchased from the fish farm S.C. Acva Prod S.R.L. Cefa, Bihor County these were obtained by artificial reproduction. After taking and selection, the fecundated spawns were introduced in 10 Nunk culture plates of 45 ml, where we introduced 40 ml water, too. We created 3 batches, with two replications, namely: batch 1 – control, batch 2 – in water, we added ethinylestradiol (EE2 in concentration of 1.5 ng L-1 and batch 3 – we added in water a concentration of 7 ng L-1 EE2. During the incubation, the Nunk plates were kept in breeding aquariums, at a temperature of 24°C. Successive to the supervision of embryos in batch 3, 48 hours post-fecundation, we could observe evolution stagnations, 70% of them being in the stage of 40 somites of the segmentation period. At the same age, 100% of the control batch- embryos entered the stage of advanced faringula, and in batch 2 all embryos were in the stage of incipient faringula. 60-72 hours post-fecundation, all embryos in the batch 3 died, 90% in the 40 somite stage of the segmentation period and 10% in the stage of incipient faringula. 85 hours post-fecundation, all embryos belonging to the control batch were in the larva stage, while in batch 2, 90% were in the larva stage and 10% died in the stage of advanced faringula.

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

Science.gov (United States)

Kelley, Rebecca L; Gardner, David K

2017-05-01

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 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 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 media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P 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. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Integrated toxic evaluation of sulfamethazine on zebrafish: Including two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure).

Science.gov (United States)

Yan, Zhengyu; Yang, Qiulian; Jiang, Weili; Lu, Jilai; Xiang, Zhongrun; Guo, Ruixin; Chen, Jianqiu

2018-03-01

Persistence of antibiotics in aquatic environment may pose a risk to the non-target aquatic organisms. This study provided an integrated evaluation to analyze the toxic stress of sulfamethazine (SMZ) on zebrafish in two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure). Zebrafish embryos and adult zebrafish were exposed to SMZ at 0.2, 20 and 2000 μg/L, respectively. The results showed that SMZ at any given concentration inhibited the hatching of embryos at 58-96 hpf (hours post-fertilization). Our result also indicated that two major kinds of the malformation, which was induced by the antibiotic, were edema and spinal curvature. Additionally, the antibiotic stimulated the heartbeat while reduced the body length of the embryo at 72 hpf. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents significantly increased at 120 hpf when the embryos were exposed to the lowest concentration (0.2 μg/L) of the antibiotic. On the other hand, the antibiotic induced SOD activities and MDA contents in adult zebrafish in the exposure and re-exposure periods. The MDA contents could recover while SOD activities still increased in 2 d after the exposure. Both SOD activities and MDA contents could recover in 7 d after the exposure. Levels of SOD and MDA in the re-exposure were higher than those in the first exposure. Our results suggested that SMZ had toxic effects on both embryos and adult zebrafish, and provided an integrated evaluation of the toxic effects of SMZ on zebrafish at a new perspective. Copyright © 2017 Elsevier Ltd. All rights reserved.

Non-invasive metabolomic profiling of embryo culture media and morphology grading to predict implantation outcome in frozen-thawed embryo transfer cycles.

Science.gov (United States)

Li, Xiong; Xu, Yan; Fu, Jing; Zhang, Wen-Bi; Liu, Su-Ying; Sun, Xiao-Xi

2015-11-01

Assessment of embryo viability is a crucial component of in vitro fertilization and currently relies largely on embryo morphology and cleavage rate. Because morphological assessment remains highly subjective, it can be unreliable in predicting embryo viability. This study investigated the metabolomic profiling of embryo culture media using near-infrared (NIR) spectroscopy for predicting the implantation potential of human embryos in frozen-thawed embryo transfer (FET) cycles. Spent embryo culture media was collected on day 4 after thawed embryo transfer (n = 621) and analysed using NIR spectroscopy. Viability scores were calculated using a predictive multivariate algorithm of fresh embryos with known pregnancy outcomes. The mean viability indices of embryos resulting in clinical pregnancy following FET were significantly higher than those of non-implanted embryos and differed between the 0, 50, and 100 % implantation groups. Notably, the 0 % group index was significantly lower than the 100 % implantation group index (-0.787 ± 0.382 vs. 1.064 ± 0.331, P  0.05). NIR metabolomic profiling of thawed embryo culture media is independent of morphology and correlates with embryo implantation potential in FET cycles. The viability score alone or in conjunction with morphologic grading is a more objective marker for implantation outcome in FET cycles than morphology alone.

Embryo sac formation and early embryo development in Agave tequilana (Asparagaceae).

Science.gov (United States)

González-Gutiérrez, Alejandra G; Gutiérrez-Mora, Antonia; Rodríguez-Garay, Benjamín

2014-01-01

Agave tequilana is an angiosperm species that belongs to the family Asparagaceae (formerly Agavaceae). Even though there is information regarding to some aspects related to the megagametogenesis of A. tequilana, this is the first report describing the complete process of megasporogenesis, megagametogenesis, the early embryo and endosperm development process in detail. The objective of this work was to study and characterize all the above processes and the distinctive morphological changes of the micropylar and chalazal extremes after fertilization in this species. The agave plant material for the present study was collected from commercial plantations in the state of Jalisco, Mexico. Ovules and immature seeds, previously fixed in FAA and kept in ethanol 70%, were stained based on a tissue clarification technique by using a Mayer's-Hematoxylin solution. The tissue clarification technique was successfully used for the characterization of the megasporogenesis, megagametogenesis, mature embryo sac formation, the early embryo and endosperm development processes by studying intact cells. The embryo sac of A. tequilana was confirmed to be of the monosporic Polygonum-type and an helobial endosperm formation. Also, the time-lapse of the developmental processes studied was recorded.

Embryo quality and impact of specific embryo characteristics on ongoing implantation in unselected embryos derived from modified natural cycle in vitro fertilization

NARCIS (Netherlands)

Pelinck, Marie-Jose; Hoek, Annemieke; Simons, Arnold H. M.; Heineman, Maas Jan; van Echten-Arends, Janny; Arts, Eus G. J. M.

Objective: To study the implantation potential of unselected embryos derived from modified natural cycle IVF according to their morphological characteristics. Design: Cohort study. Setting: Academic department of reproductive medicine. Patient(S): A series of 449 single embryo transfers derived from

Methanol as a cryoprotectant for equine embryos.

Science.gov (United States)

Bass, L D; Denniston, D J; Maclellan, L J; McCue, P M; Seidel, G E; Squires, E L

2004-09-15

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.

DNA-repair after UV-irradiation in skin fibroblasts from patients with actinic keratosis

International Nuclear Information System (INIS)

Sbano, E.; Andreassi, L.; Fimiani, M.; Valentino, A.; Baiocchi, R.

1978-01-01

Autoradiographic counting technique was utilized to measure the ultraviolet-induced unscheduled DNA synthesis of skin fibroblasts from 12 patients with chronic actinic keratosis and from 12 healthy donors of about the same age. In order to reveal a possible regional difference of DNA repair between the parts of the body ordinarily exposed and those parts unexposed to sunlight, two cell strains were used for each examined subject; one developed from the forehead skin and the other from the abdominal or axillary skin. Unscheduled DNA synthesis appeared depressed in actinic keratosis patients, as compared with controls. In all examined subjects, however, cell strains from exposed skin showed a DNA repair more active than cell strains from unexposed skin. These findings show that skin cancer may be promoted in actinic keratosis patients by a defect of DNA repair. The exalted DNA repair of chronically sun exposed skin is probably the consequence of a defensive process caused by enzymatic induction. (orig.) [de

Identification of differentially expressed genes in sexed pig embryos during post-hatching development in primiparous sows exposed to differing intermittent suckling and breeding strategies

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Stephen Tsoi

2016-09-01

Full Text Available The aim of commercial pig breeding programs is to maximize the number of pigs produced per sow per year. Given that sows exhibit an estrus during lactation is a potential means of increasing productivity of a pig breeding herd without reducing in lactation length, conventionally, weaning of piglets at a relatively young age is often related to post-weaning piglet performance which compromises piglet welfare. Therefore, intermittent suckling (IS is a management technique in which lactating sows are separated from their piglets for a fixed period of the days and allowing sows to continue nursing piglets while exhibiting estrus and being breed during lactation, thereby promoting both piglet well-being and sow reproductive performance [1]. For this study, primiparous sows (PP were exposed to 28 day (D28 lactation with intermittent suckling (IS during the final week prior to weaning. The sows detected to be in estrus during lactation were either bred at this first estrus (FE during lactation (IS21FE, or were “skipped” and bred at their second estrus which occurred after final weaning at D28 (IS21SE. Despite the benefits of IS, the effects of the maternal physiology related to breeding during lactation on embryonic transcriptome are largely unknown. Recent advances in the ability to assess embryonic gene expression in both sexes have made these analyses possible. Here, we describe the experimental procedures of two color microarray analyses and annotation of differentially expressed (DE genes in detail corresponding to data deposited at NCBI in the Gene Expression Omnibus under accession number GSE53576 and GSE73020 for day 9 embryos (D9E and day 30 embryos (D30E respectively. Although only a few DE genes were discovered between IS21FE and IS21SE in both sexes from D9E or D30E, the raw data are still valuable for future use to understand the gene expression profiling from two different developmental stages.

Non-invasive analysis of bovine embryo metabolites during in vitro embryo culture using nuclear magnetic resonance

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Marcello Rubessa

2016-12-01

Full Text Available The ability to identify embryos that have the highest developmental potential from a cohort would significantly increase the chances of achieving pregnancy. Metabolic analysis is a well-established analytical approach in biological systems. Starting from this idea, we chose to use high-resolution nuclear magnetic resonance (1H-NMR spectroscopy. The aim of this study was to determine if it is possible to select viable embryos after 48 h of culture using metabolic activity as the parameter. We evaluated embryo metabolism after the first 48 h of culture and compared the activity of cleaved embryos that became blastocysts to cleaved embryos that did not develop to blastocysts, and in vitro fertilized (IVF blastocysts and parthenogenetic-activated (PA blastocysts. Our results show that citrate, pyruvate, myo-inositol and lysine have great impact on predicting embryo development. When we compared IVF and PA blastocysts, we found that acetate and phenylalanine concentrations are excellent parameters for evaluating blastocyst quality. Combining all these results, we were able to create a formula that predicts zygote development after 2 days of culture. In conclusion, we found that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1H-NMR.

Functions and Mechanisms of Fibroblast Growth Factor (FGF Signalling in Drosophila melanogaster

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Hans-Arno J. Müller

2013-03-01

Full Text Available Intercellular signalling via growth factors plays an important role in controlling cell differentiation and cell movements during the development of multicellular animals. Fibroblast Growth Factor (FGF signalling induces changes in cellular behaviour allowing cells in the embryo to move, to survive, to divide or to differentiate. Several examples argue that FGF signalling is used in multi-step morphogenetic processes to achieve and maintain a transitional state of the cells required for the control of cell fate. In the genetic model Drosophila melanogaster, FGF signalling via the receptor tyrosine kinases Heartless (Htl and Breathless (Btl is particularly well studied. These FGF receptors affect gene expression, cell shape and cell–cell interactions during mesoderm layer formation, caudal visceral muscle (CVM formation, tracheal morphogenesis and glia differentiation. Here, we will address the current knowledge of the biological functions of FGF signalling in the fly on the tissue, at a cellular and molecular level.

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

Science.gov (United States)

Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

2015-08-15

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

Fibroblast cultures in duchenne muscular dystrophy

International Nuclear Information System (INIS)

Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

1977-01-01

Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

Improving embryo quality in assisted reproduction

NARCIS (Netherlands)

Mantikou, E.

2013-01-01

The goal of this thesis was to improve embryo quality in assisted reproductive technologies by gaining more insight into human preimplantation embryo development and by improving in vitro culture conditions. To do so, we investigated an intriguing feature of the human preimplantation embryo, i.e.

Reproductive function in mice exposed to ancestral and direct irradiation

International Nuclear Information System (INIS)

Nash, D.J.; Sprackling, L.S.

1978-01-01

Reproduction was studied in 13 inbred strains of mice that had been exposed continuously to 60 Co gamma radiation for varying numbers of generations. At weaning the mice were removed from the irradiation chamber and were tested for reproductive performance. Ancestral and direct levels of irradiation were determined for each animal. Each irradiated or control female was scored as fertile or sterile, and in utero litter counts were made in pregnant females that were dissected past the 10th day of pregnancy. The number of resorptions, dead embryos, and live embryos were counted, and the ratio of living embryos to the total number of embryos was determined for each litter. The overall fertility curves were sigmoid in the range of doses below those which caused complete sterility, which indicated some sort of cumulative damage. In 11 of the 13 strains studied, an increase in ancestral and/or direct irradiation led to significant decreases in fertility. The means of the number alive in the litters for the control and irradiated mice in each strain showed a definite trend toward fewer live mice in utero after irradiation. Least-squares analyses of variance were made to detect possible effects of any of six irradiation variables (ancestral linear, ancestral quadratic, ancestral cubic, direct linear, direct quadratic, or direct cubic) or of strain differences on total litter size and on ratio. Strain effects were significant in each instance. Litter size was more likely to be affected by radiation variables than ratios were

Evidence against a direct role for oxidative stress in cadmium-induced axial malformation in the chick embryo

International Nuclear Information System (INIS)

Thompson, Jennifer; Doi, Takashi; Power, Eoin; Balasubramanian, Ishwarya; Puri, Prem; Bannigan, John

2010-01-01

Cadmium (Cd) is a powerful inducer of oxidative stress. It also causes ventral body wall defects in chick embryos treated at Hamburger-Hamilton stages 16-17. By measuring malondialdehyde levels (TBARS method) and cotreating with antioxidants (tempol, ascorbate, and N-acetylcysteine), we sought to determine if oxidative stress were directly related to teratogenesis. We also investigated the expression of mRNAs for antioxidant enzymes superoxide dismutase (SOD) -1 and -2, catalase (CAT), and glutathione peroxidase (GPx). RT-PCR showed reductions in SOD-1, SOD-2, and CAT 1 hour after treatment with Cd. MDA levels increased 4 hours after Cd, and remained elevated 24 hours after treatment. Of the antioxidants, only N-acetylcysteine reduced MDA levels to control values. Nonetheless, no antioxidant could reduce embryo lethality or malformation rates. Furthermore, MDA levels 24 hours after treatment were identical in malformed and normal embryos exposed to Cd. Hence, we conclude that oxidative stress may not have a direct role in Cd teratogenesis.

Adaptive response to ionising radiation induced by cadmium in zebrafish embryos

International Nuclear Information System (INIS)

Choi, V W Y; Ng, C Y P; Kong, M K Y; Yu, K N; Cheng, S H

2013-01-01

An adaptive response is a biological response where the exposure of cells or animals to a low priming exposure induces mechanisms that protect the cells or animals against the detrimental effects of a subsequent larger challenging exposure. In realistic environmental situations, living organisms can be exposed to a mixture of stressors, and the resultant effects due to such exposures are referred to as multiple stressor effects. In the present work we demonstrated, via quantification of apoptosis in the embryos, that embryos of the zebrafish (Danio rerio) subjected to a priming exposure provided by one environmental stressor (cadmium in micromolar concentrations) could undergo an adaptive response against a subsequent challenging exposure provided by another environmental stressor (alpha particles). We concluded that zebrafish embryos treated with 1 to 10 μM Cd at 5 h postfertilisation (hpf) for both 1 and 5 h could undergo an adaptive response against subsequent ∼4.4 mGy alpha-particle irradiation at 10 hpf, which could be interpreted as an antagonistic multiple stressor effect between Cd and ionising radiation. The zebrafish has become a popular vertebrate model for studying the in vivo response to ionising radiation. As such, our results suggested that multiple stressor effects should be carefully considered for human radiation risk assessment since the risk may be perturbed by another environmental stressor such as a heavy metal. (paper)

Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program.

Science.gov (United States)

Jacob, J C F; Haag, K T; Santos, G O; Oliveira, J P; Gastal, M O; Gastal, E L

2012-04-01

In the present study, 809 uterine flushes and 454 embryo transfers performed in mares over a 4-yr interval were examined to evaluate the effects of: (1) the day of embryo collection on recovery rates; (2) the degree of synchrony between donor and recipient mares on pregnancy rates; (3) the recipient day post ovulation on pregnancy rates; and (4) the age of the embryo at recovery on pregnancy rates at 60 days. Uterine flushes were performed on Days 6, 7, 8, 9, and 10 (Day 0 = ovulation) and embryos were transferred to recipients with degrees of synchrony varying between +1 to -6 (recipient ovulated 1 day before through 6 days after the donor). Recipient mares ranged from 2 to 8 days post ovulation. Embryo recovery rates were similar for flushes performed on Day 7 (61%), Day 8 (66%), Day 9 (59%), and Day 10 (56%), but the embryo recovery rate was lower (P recipient mares on Day 2 (33%) compared with mares on Day 3 (66%), Day 4 (66%), Day 5 (62%), Day 6 (55%), Day 7 (58%), and Day 8 (56%). Pregnancy rate was higher (P recipient mares does not need to be as restricted as previously reported in horses. Acceptable pregnancy rates (e.g., 70%, 99/142) were obtained even when recipient mares ovulated 4 to 5 days after the donors; (3) similar pregnancy rates were obtained when recipient mares received embryos within a large range of days post ovulation (Days 3 to 8); and (4) Day 7 embryos produced higher pregnancy rates when compared with Days 8 and 9 embryos. In clinical terms, the application of these new findings will be beneficial to large equine embryo transfer operations in producing more pregnancies per season. Copyright © 2012 Elsevier Inc. All rights reserved.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study.

Science.gov (United States)

Geber, Selmo; Bossi, Renata; Lisboa, Cintia B; Valle, Marcelo; Sampaio, Marcos

2011-04-28

We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

Laser confers less embryo exposure than acid tyrode for embryo biopsy in preimplantation genetic diagnosis cycles: a randomized study

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Valle Marcelo

2011-04-01

Full Text Available Abstract We compared two methods of zona pellucida drilling. 213 embryos were biopsied with acid Tyrode. Each biopsy took 3 minutes and the entire procedure ~29 minutes. 5% of blastomeres lysed, 49% of embryos became blastocyst and 36% of patients became pregnant. 229 embryos were biopsied with laser. Each biopsy took 30 seconds and the entire procedure ~7 minutes. 2.5% of blastomeres lysed, 50.6% of embryos became blastocyst and 47% of patients became pregnant. We can conclude that laser can be used for embryo biopsy. Reduction of embryo exposure and of removed blastomeres is associated with increased blastocysts available for transfer and a better clinical outcome.

mRNA expression of genes involved in inflammation and haemostasis in equine fibroblast-like synoviocytes following exposure to lipopolysaccharide, fibrinogen and thrombin

DEFF Research Database (Denmark)

Andreassen, Stine Mandrup; Berg, Lise Charlotte; Nielsen, Søren Saxmose

2015-01-01

Background: Studies in humans have shown that haemostatic and inflammatory pathways both play important roles in the pathogenesis of joint disease. The aim of this study was to assess mRNA expression of haemostatic and inflammatory factors in cultured equine fibroblast-like synoviocytes exposed t...

Biochemical changes to fibroblast cells subjected to ionizing radiation.

Science.gov (United States)

Jones, Pamala; Benghuzzi, Hamed; Tucci, Michelle; Richards, Latoya; Harrison, George; Patel, Ramesh

2008-01-01

High energy X-rays are capable of interacting with biological membranes to cause both functional and structural modifications. The goal of the present study was to investigate the effects human fibroblast cells exposed multiple times to 10 Gy over time. Following exposures of 2, 3, or 4 times to 10 Gy/10min the cells were evaluated for cell number changes, membrane damage, and intracellular glutathione content after 24, 48 and 72 hours. Twenty-four hours following exposure the cell numbers were reduced and increased levels of cellular membrane damage was evident. This trend was observed for the duration of the study. Interestingly, there was not an exposure dependent increase in cell damage or cell loss with time. Intracellular antioxidant systems were activated as indicated by anincrease in total cellular glutathione content. Additional studies are needed to determine if the cellular reduction is caused by a direct effect of the X-rays targeting the DNA or an indirect effect of the X-ray targeting the cellular membrane, which then generates radicals that target cell cycle checkpoints or DNA damage. In conclusion, fibroblast cells can be used to determine early and late events of cellular function following exposure to harmful levels of radiation exposure and results of exposure can be seen within twenty four hours.

Trichomonas vaginalis and Tritrichomonas foetus: interaction with fibroblasts and muscle cells - new insights into parasite-mediated host cell cytotoxicity

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Ricardo Chaves Vilela

2012-09-01

Full Text Available Trichomonas vaginalis and Tritrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that trichomonas infection of the connective and muscle tissues is likely to occur; such

The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

Science.gov (United States)

Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

2017-04-06

The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

DEFF Research Database (Denmark)

Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

1992-01-01

major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

Change in the radiation responses of oogonia in the embryos and fry of the fish Oryzias latipes

International Nuclear Information System (INIS)

Hamaguchi, S.

1976-01-01

Embryos of the fish Oryzias latipes were irradiated with 1000 R of X-rays 1 day before hatching, and the post-irradiation change in the female germ-cell population was observed. Scarcely any reduction in the number of oogonia was observed, but their proliferation was inhibited. Repopulation occurred between 12 and 20 days after hatching. These responses were quite different from those of germ cells in the irradiated fry. Embryos and/or fry were also exposed to 1000 R of X-rays 1 day before hatching and 0, 1, 2, and 3 days after hatching. A comparison of their responses suggested that the change in the radiation responses of oogonia is correlated with the initiation of meiosis. (author)

Psychological study of in vitro fertilization-embryo transfer participants' attitudes toward the destiny of their supernumerary embryos.

Science.gov (United States)

Laruelle, C; Englert, Y

1995-05-01

To study the motivations underlying IVF-ET participants' choice to donate or destroy their supernumerary embryos. Couples' opinions are studied through a questionnaire and a psychological interview. Two hundred couples about to undergo IVF-ET. The fertility unit of an academic hospital. Couples' choice for supernumerary embryos' destiny; opinions on embryo status, on importance of genetic lineage in the filial bonding, on gamete donation, and on multiple pregnancy risk. Donation is the most frequent choice but destruction is tolerated by almost all the couples (92%). Couples considering the embryo as a child choose destruction as frequently as donation but refuse experimentation on the embryo. Donation is highest among couples who stress education more than genetic lineage in parental bonding. This is confirmed by the choice of the couples requiring donor gametes. Couples express differing attitudes toward risks of twins and risks of triplets: twins are much more desired than triplets, which are frequently refused. Couples' opinions on the respective importance of genetic lineage and education in defining parental bonding are more determinant in their decision to destroy or to donate their supernumerary embryos than their opinions on the in vitro embryo status, which only determines their attitude toward experimentation.

Mechanistic dissection of plant embryo initiation

NARCIS (Netherlands)

Radoeva, T.M.

2016-01-01

Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in

Ethanol vapour induced dilated cardiomyopathy in chick embryos

International Nuclear Information System (INIS)

Kamran, K.; Khan, M.Y.; Minhas, L.A.

2013-01-01

Objective: To study the effects of ethanol vapour inhalation on the heart chambers of chick embryo. Methods: The case-control study was conducted at the College of Physicians and Surgeons Pakistan regional centre in Islamabad from January to October 2007. Both experimental and control groups were divided into three sub-groups each, based on the day of the sacrifice. Each group was dissected on day 7, day 10 and day 22 or hatching whichever was earlier. The experimental sub-groups sacrificed on day 7, day 10 and on hatching, were exposed to ethanol vapours till day 6, 9 and 9 of incubation respectively. The diameter of all 4 chambers was measured in experimental hearts and compared with age-matched controls. SPSS 10 was used for statistical analysis. Results: Ethanol vapour exposure caused widening of all heart chambers in the experimental chick embryos sacrificed on day 7 and day 10 compared to the controls. The chambers of newly hatched chick hearts showed dilatation in all the chambers except the left ventricle. Conclusion: Ethanol vapour exposure during development affects the heart, resulting in the widening of all heart chambers. The exposure is as dangerous as drinking alcohol. Alcohol vapour exposure during development leads to progressive dilatation in different heart chambers, producing dilated cardiomyopathy. (author)

UVB radiation prevents skeleton growth and stimulates the expression of stress markers in sea urchin embryos

International Nuclear Information System (INIS)

Bonaventura, Rosa; Poma, Veronica; Costa, Caterina; Matranga, Valeria

2005-01-01

Ozone depletion results in an increased flux of biologically damaging radiations reaching the earth. Although ultraviolet (UV) penetration is attenuated by the seawater, harmful effects can be still observed at low depths where sea urchin embryos are living. We have used Paracentrotus lividus embryos to study the impacts of UV radiation on their development. Blastula cultures were exposed to different doses of UVB (312 nm) radiations and the resulting endpoint effects were evaluated in terms of embryonic morphological abnormalities, variations in specific gene expression, and changes in the levels of stress proteins. We found that embryos were moderately sensitive to 50 J/m 2 UVB radiation; an increase in the number of developmentally delayed and malformed embryos was detected when increasing doses, up to 1000 J/m 2 , were used. Major developmental defects, observed 24 and 48 h after exposure, consisted in the failure of skeleton elongation and patterning. Accordingly, we found a reduction in the number of primary mesenchyme cells that expressed Pl-SM30, a gene coding for one of the specific matrix proteins of the skeleton. The morphological effects observed 1, 24, and 48 h after exposure were correlated with a dose-dependent increase in the level and in the activation of two recognized stress markers, namely hsp70 and p38 MAPk, respectively, consistent with their role in mediating cellular response to stress and suggesting a function in embryo survival

A descriptive study to provide evidence of the teratogenic and cellular effects of sibutramine and ephedrine on cardiac- and liver-tissue of chick embryos.

Science.gov (United States)

Oberholzer, Hester Magdalena; Van Der Schoor, Ciska; Taute, Helena; Bester, Megan Jean

2015-08-01

Exposure to drugs during pregnancy is a major concern, as some teratogenic compounds can influence normal foetal development. Although the use of drugs during pregnancy should generally be avoided, exposure of the developing foetus to teratogens may occur unknowingly since these compounds may be hidden in products that are being marketed as "all natural." The aim of the current study was to investigate the possible teratogenic and cellular effects of sibutramine-a serotonin-norepinephrine reuptake inhibitor used in the treatment of obesity-on the heart and liver tissue of chick embryos. Ephedrine was used as a positive control. The chick embryo model was chosen because it has been used in studying developmental and experimental biology and teratology with great success. The embryos were exposed to three different concentrations of sibutramine and ephedrine respectively. The results obtained revealed that both compounds exhibited embryotoxicity when compared to the control groups. Liver and heart tissue of the exposed embryos was severely affected by these compounds in a dose-related manner. Morphology similar to that of muscle dystrophy was observed in the heart, where the muscle tissue was infiltrated by adipose and connective tissue. Severe liver steatosis was also noted. A more in-depth investigation into the molecular pathways involved might provide more information on the exact mechanism of toxicity of these products influencing embryonic development. © 2015 Wiley Periodicals, Inc.

The First Human Cloned Embryo.

Science.gov (United States)

Cibelli, Jose B.; Lanza, Robert P.; West, Michael D.; Ezzell, Carol

2002-01-01

Describes a process known as parthenogenesis which produces cloned, early-stage embryos and human embryos generated only from eggs. Speculates that this technology puts therapeutic cloning within reach. (DDR)

Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

Directory of Open Access Journals (Sweden)

Ingrid R. Cordeiro

2015-09-01

Full Text Available Human adipose-derived stromal cells (hADSC are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1 regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues.

Impact of matrix stiffness on fibroblast function

Energy Technology Data Exchange (ETDEWEB)

El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

2017-05-01

Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

Hatchability and survival of oncomiracidia of Paradiplozoon ichthyoxanthon (Monogenea: Diplozoidae) exposed to aqueous aluminium.

Science.gov (United States)

Gilbert, Beric M; Avenant-Oldewage, Annemariè

2016-07-28

Monogenea is a diverse group of ectoparasites showing great potential as sentinel organisms for monitoring environmental health. Exposure to metals negatively affects infrapopulations of monogeneans and exposure to aluminium has been found to negatively impact the survival of gyrodactylids. Samples of infected host fish, the smallmouth yellowfish Labeobarbus aeneus (Cyprinidae), were collected from the Vaal Dam, South Africa and transported back to the laboratory in dark 160 l containers. Eggs of the monogenean Paradiplozoon ichthyoxanthon infecting L. aeneus were collected and exposed to varying concentrations of aluminium along with a control group in static tanks. The eggs were checked every 24 h and hatching commenced 13-14 days after exposure. Water samples were taken from exposure tanks and acidified for analysis of Al levels with inductively-coupled plasma mass spectrometry. Hatching of eggs was variable between exposures, and in 30 μg Al/l and 60 μg Al/l was found to occur before eggs in control beakers, whereas, exposure to 120 μg Al/l delayed hatching and reduced hatchability. Survival of hatched oncomiracidia was concentration dependent and negatively correlated with aluminium concentrations. Lowest survival was recorded for 60 μg Al/l and 120 μg Al/l where all larvae died shortly after or during hatching. Normal development of embryos of P. ichthyoxanthon within eggs exposed to all doses of aluminium indicates that the egg shell is moderately impermeable to metals and inhibits movement of aluminium across the shell and interacting with developing embryos. Higher larval mortality rate in 120 μg/l exposure can be related to aluminium crossing the egg shell in the late stages and causing death of unhatched yet fully developed embryos, possibly due to changes in the permeability of the egg shell as embryos neared developmental completion. Accelerated death of oncomiracidia after hatching indicates sensitivity toward high concentrations of aluminium.

Effect of culture medium volume and embryo density on early mouse embryonic development: tracking the development of the individual embryo.

Science.gov (United States)

Dai, Shan-Jun; Xu, Chang-Long; Wang, Jeffrey; Sun, Ying-Pu; Chian, Ri-Cheng

2012-07-01

To determine the optimal volume or density of embryos for the well-of-the-well (WOW) system in order to track the development of individual embryos and to determine whether the WOW system can reverse the negative impact of culturing embryos singly. (1) Mouse embryos (groups of nine at the 2-cell stage) were cultured in 6.25 μl, 12.50 μl, 25.00 μl and 50.00 μl of droplets of culture medium under paraffin oil; (2) Groups of three, six, nine and twelve embryos at the 2-cell stage were cultured in 50 μl of droplet of culture medium under paraffin oil; (3) Groups of nine embryos at the 2-cell stage were cultured in 50 μl of droplet under paraffin oil with or without nine micro-wells made on the bottom of the Petri dish into each of which were placed one of the nine embryos (WOW system). Also single 2-cell stage embryos was cultured individually in 5.5 μl of droplet of culture medium under paraffin oil with or without a single micro-well made on the bottom of the Petri dish (WOW system for single culture). At the end of culture, the percentages of blastocyst development, hatching and hatched blastocysts were compared in each group. The blastocysts were fixed for differential staining. The blastocyst development was significantly higher (P WOW system. The blastocyst development was not improved when single embryo cultured individually in a micro-well was compared to single embryo cultured individually without micro-well. The total cell numbers of blastocysts were significantly higher in group embryo culture than single embryo culture regardless of whether the WOW system was used. In addition, the total cell numbers of blastocysts were significantly higher (P WOW system than without. Group embryo culture is superior to single embryo culture for blastocyst development. The WOW system with 50 μl of droplet of culture medium can be used to track the individual development of embryo cultured in groups while preserving good embryonic development. The reduced

Trichostatin A (TSA) improves the development of rabbit-rabbit intraspecies cloned embryos, but not rabbit-human interspecies cloned embryos.

Science.gov (United States)

Shi, Li-Hong; Miao, Yi-Liang; Ouyang, Ying-Chun; Huang, Jun-Cheng; Lei, Zi-Li; Yang, Ji-Wen; Han, Zhi-Ming; Song, Xiang-Fen; Sun, Qing-Yuan; Chen, Da-Yuan

2008-03-01

The interspecies somatic cell nuclear transfer (iSCNT) technique for therapeutic cloning gives great promise for treatment of many human diseases. However, the incomplete nuclear reprogramming and the low blastocyst rate of iSCNT are still big problems. Herein, we observed the effect of TSA on the development of rabbit-rabbit intraspecies and rabbit-human interspecies cloned embryos. After treatment with TSA for 6 hr during activation, we found that the blastocyst rate of rabbit-rabbit cloned embryos was more than two times higher than that of untreated embryos; however, the blastocyst rate of TSA-treated rabbit-human interspecies cloned embryos decreased. We also found evident time-dependent histone deacetylation-reacetylation changes in rabbit-rabbit cloned embryos, but not in rabbit-human cloned embryos from fusion to 6 hr after activation. Our results suggest that TSA-treatment does not improve blastocyst development of rabbit-human iSCNT embryos and that abnormal histone deacetylation-reacetylation changes in iSCNT embryos may account for their poor blastocyst development. (c) 2008 Wiley-Liss, Inc.

Influence of dose rate on the transformation of Syrian hamster embryo cells by fission-spectrum neutrons

Energy Technology Data Exchange (ETDEWEB)

Jones, C.A.; Sedita, B.A.; Hill, C.K.; Elkind, M.M.

1988-01-01

Several explanations for this neutron dose-rate effect have been proposed, but further investigation is necessary to determine the mechanisms involved. In all cell transformation studies to date the immortalized, aneuploid 10T1/2 cell-line has been used. These cells may be premalignant; thus their response characteristics and, in particular, the nature of the transformation event, might differ from that in a normal, fibroblast cell. One reason for the present study was to determine whether the low-dose-rate effect of fission neutrons could be demonstrated in normal cells. If so, a normal cell system, which would more closely resemble a normal in vivo system, could be used for mechanistic studies. We chose Syrian hamster embryo (SHE) fibroblasts which are normal, diploid cells with a limited life span in culture. Upon exposure to low doses of ionizing radiation, the fraction of the cells that are transformed can be identified in a standard 8--10 day colony assay by examining their clonal morphology. Transformed cells form colonies with a dense, criss-crossed or piled-up structure. A high percentage of the transformed colonies can be further propagated and will acquire additional neoplastic characteristics; i.e., anchorage independence, immortality, altered proteolytic activity, karyotype alterations, and finally, tumorigenicity.

Influence of dose rate on the transformation of Syrian hamster embryo cells by fission-spectrum neutrons

International Nuclear Information System (INIS)

Jones, C.A.; Sedita, B.A.; Hill, C.K.; Elkind, M.M.

1988-01-01

Several explanations for this neutron dose-rate effect have been proposed, but further investigation is necessary to determine the mechanisms involved. In all cell transformation studies to date the immortalized, aneuploid 10T1/2 cell-line has been used. These cells may be premalignant; thus their response characteristics and, in particular, the nature of the transformation event, might differ from that in a normal, fibroblast cell. One reason for the present study was to determine whether the low-dose-rate effect of fission neutrons could be demonstrated in normal cells. If so, a normal cell system, which would more closely resemble a normal in vivo system, could be used for mechanistic studies. We chose Syrian hamster embryo (SHE) fibroblasts which are normal, diploid cells with a limited life span in culture. Upon exposure to low doses of ionizing radiation, the fraction of the cells that are transformed can be identified in a standard 8--10 day colony assay by examining their clonal morphology. Transformed cells form colonies with a dense, criss-crossed or piled-up structure. A high percentage of the transformed colonies can be further propagated and will acquire additional neoplastic characteristics; i.e., anchorage independence, immortality, altered proteolytic activity, karyotype alterations, and finally, tumorigenicity

Noninvasive metabolomic profiling as an adjunct to morphology for noninvasive embryo assessment in women undergoing single embryo transfer

NARCIS (Netherlands)

Seli, E.; Vergouw, C.G.; Morita, H.; Botros, L.; Roos, P.; Lambalk, C.B.; Yamashita, N.; Kato, O.; Sakkas, D.

2010-01-01

Objective: To determine whether metabolomic profiling of spent embryo culture media correlates with reproductive potential of human embryos. Design: Retrospective study. Setting: Academic and a private assisted reproductive technology (ART) programs. Patient(s): Women undergoing single embryo

Die Behandlung menschliches Embryos und Menschenwurde

OpenAIRE

Matsui, Fumio

2002-01-01

We are confronted with an old and new problem, which has come up with the progress of modern biotechnologies: what is a life or when does a life begin? The expectation of order-made medicine has build up since the discovery of Embryo Stem cell called "a dream master cell", while there is any condemnation against the destruction of human embryo in order to gain it. It is a question whether a human embryo is a human being in the world. Human dignity(=HD) is a principle that keeps human embryos ...

Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate.

Science.gov (United States)

Han, Zhihua; Wang, Qiangwei; Fu, Jie; Chen, Hongshan; Zhao, Ye; Zhou, Bingsheng; Gong, Zhiyuan; Wei, Si; Li, Jun; Liu, Hongling; Zhang, Xiaowei; Liu, Chunsheng; Yu, Hongxia

2014-05-01