scholarly journals Direct Visualization of Live Zebrafish Glycan via Single-step Metabolic Labeling with Fluorophore-tagged Nucleotide Sugars

2019 ◽  
Author(s):  
Senlian Hong ◽  
Pankaj Sahai-Hernandez ◽  
David Traver ◽  
Peng Wu
Keyword(s):  
Developmental Stages ◽  
Single Step ◽  
Zebrafish Embryos ◽  
Metabolic Labeling ◽  
Cell Stage ◽  
Imaging Probes ◽  
Nucleotide Sugars ◽  
Living Organisms ◽  
The One

ABSTRACTDynamic turnover of cell-surface glycans is involved in a myriad of biological events, making this process an attractive target for in vivo molecular imaging. The metabolic glycan labeling coupled with ‘bioorthogonal chemistry’ has paved the way for visulizing glycans in living organisms. However, a two-step labeling sequence is required, which is prone to tissue penetration difficulties of the imaging probes. Here, by exploring the substrate promiscuity of endogenous glycosyltransferases, we developed a single-step fluorescent glycan labeling strategy by using fluorophore-tagged analogs of nucleotide sugars directly. Injecting the fluorophore-tagged sialic acid and fucose into the yolk of zebrafish embryos at the one-cell stage enables a systematic imaging of sialylation and fucosylation in live zebrafish embryos at various developmental stages. From these studies, we obtained insights into the role of sialylated and fucosylated glycans in zebrafish hematopoiesis.

scholarly journals Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos

Reproduction ◽  
2004 ◽  
Vol 128 (3) ◽  
pp. 301-311 ◽  
Author(s):  
Paolo Rinaudo ◽  
Richard M Schultz
Keyword(s):  
Gene Expression ◽  
Preimplantation Embryos ◽  
Expression Analysis Systematic Explorer ◽  
Cell Stage ◽  
Global Pattern ◽  
Preimplantation Mouse Embryos ◽  
Preimplantation Mouse ◽  
Global Patterns ◽  
The One

Culture of preimplantation embryos affects gene expression. The magnitude of the effect on the global pattern of gene expression, however, is not known. We compared global patterns of gene expression in blastocysts cultured from the one-cell stage in either Whitten’s medium or KSOM + amino acids (KSOM/AA) with that of blastocysts that developed in vivo, using the Affymetrix MOE430A chip. The analysis revealed that expression of 114 genes was affected after culture in Whitten’s medium, whereas only 29 genes were mis-expressed after culture in KSOM/AA. Expression Analysis Systematic Explorer was used to identify biological and molecular processes that are perturbed after culture and indicated that genes involved in protein synthesis, cell proliferation and transporter function were down-regulated after culture in Whitten’s medium. A common set of genes involved in transporter function was also down-regulated after culture in KSOM/AA. These results provide insights as to why embryos develop better in KSOM/AA than in Whitten’s medium, and highlight the power of microarray analysis to assess global patterns of gene expression.

Direct Visualization of Live Zebrafish Glycans via Single‐Step Metabolic Labeling with Fluorophore‐Tagged Nucleotide Sugars

2019 ◽  
Vol 131 (40) ◽  
pp. 14465-14471 ◽  
Author(s):  
Senlian Hong ◽  
Pankaj Sahai‐Hernandez ◽  
Digantkumar Gopaldas Chapla ◽  
Kelley W. Moremen ◽  
David Traver ◽  
...  
Keyword(s):  
Single Step ◽  
Metabolic Labeling ◽  
Direct Visualization ◽  
Nucleotide Sugars

98 SUCCESSFUL PREGNANCIES FOLLOWING TRANSFER OF VITRIFIED PORCINE EMBRYOS DERIVED FROM IN VITRO-MATURED OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 157 ◽  
Author(s):  
K. Hiruma ◽  
H. Ueda ◽  
H. Saito ◽  
C. Tanaka ◽  
N. Maeda ◽  
...  
Keyword(s):  
Calf Serum ◽  
Developmental Competence ◽  
Penicillin G ◽  
Cell Stage ◽  
Epidermal Growth ◽  
Potassium Penicillin ◽  
The One ◽  
Parthenogenetic Embryos

To date only in vivo-produced embryos have successfully produced live piglets after cryopreservation. In this study, we aimed to produce piglets from vitrified embryos derived from in vitro matured (IVM) oocytes. Cumulus-oocyte complexes collected from ovaries obtained at a local slaughterhouse were matured for 44 to 45 h in NCSU23 MEDIUM supplemented with 0.6 mM cysteine, 10 ng/mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 75 �g/mL potassium penicillin G, 50 �g/mL streptomycin sulfate, and 10 IU/mL eCG/ hCG. These IVM oocytes were either activated for parthenogenesis or in vitro-fertilized (IVF). For IVF, oocytes were incubated with 5 � 106/mL of cryopreserved epididymal sperm in PGM-tac medium (Yoshioka et al. 2003 Biol. Reprod. 69, 2092-2099) for 20 h. Embryos were treated for removal of cytoplasmic lipid droplets (delipation; Nagashima et al. 1995 Nature 374, 416) at the 4- to 8-cell stages, around 50 to 54 h after activation or insemination. After culture in NCSU23 for 15 h, they were vitrified by the minimum volume cooling (MVC) method. Embryos were equilibrated with equilibration solution containing 7.5% (v/v) ethylene glycol (EG), 7.5% (v/v) dimethylsulfoxide (DMSO), and 20% (v/v) calf serum for 4 min, followed by exposure to vitrification solution containing 15% EG, 15% DMSO, 0.5 M sucrose, and 20% calf serum. Embryos were then loaded onto a Cryotop (Kitazato Supply Co., Tokyo, Japan) and immediately plunged into liquid nitrogen. Vitrified embryos were examined for viability in vitro and in vivo after warming. Their in vitro developmental competence was compared to that of corresponding control (nonvitrified) embryos. Vitrified 4- to 8-cell stage embryos, both parthenogenetic and IVF, showed developmental competence into blastocysts comparable to that of control embryos (parthenogenetic: 46.8%, 36/77 vs. 51.7%, 31/60; IVF: 40.0%, 30/75 vs. 44.3%, 35/79). Of four surrogate gilts that received a total of 251 vitrified parthenogenetic embryos, three became pregnant and had 20 fetuses (8.0%, 22 to 23 days old). Three surrogates gilts that received 267 vitrified IVF embryos all became pregnant. Of those, the one that received 47 embryos was confirmed to have eight fetuses (17.0%, 22 days old) by autopsy. The other two were examined by ultrasonography at 56 and 95 days of gestation and found to be pregnant. These results suggest that porcine embryos derived from IVM oocytes have a potential to develop into live offspring after delipation and MVC vitrification. This study was supported by PROBRAIN.

180 IDENTIFICATION AND QUANTIFICATION OF DIFFERENTIALLY REPRESENTED TRANSCRIPTS IN PREIMPLANTATION BOVINE EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 169 ◽  
Author(s):  
C. E. McHughes ◽  
G. K. Springer ◽  
L. D. Spate ◽  
R. Li ◽  
R. J. Woods ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Nuclear Transfer ◽  
Developmental Stages ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Production Techniques

Identification of transcripts that are present at key development stages of preimplantation embryos is critical for a better understanding of early embryogenesis. To that end, this project had two goals. The first was to characterize the relative abundance of multiple transcripts during several developmental stages, including metaphase II-stage oocytes (MPII), and 2-cell-stage (2-cell), precompact morula (PCM), and in vitro-produced blastocyst-stage (IVTBL) embryos. The second was to characterize differences in the relative abundance of transcripts present in in vivo- (IVVBL), in vitro-, and nuclear transfer-produced (NTBL) blastocysts. It was our hypothesis that the identification of differentially represented transcripts from these stages would reveal not only developmentally important genes, but also genes that might be aberrantly expressed due to embryo production techniques. Individual clusters from a large bovine EST project (http://genome.rnet.missouri.edu/Bovine/), which focused on female reproductive tissues and embryos, were compared using Fisher's exact test weighted by number of transcripts per tissue by gene (SAS PROC FREQ; SAS Institute, Inc., Cary, NC, USA). Of the 3144 transcripts that were present during embryogenesis, 125 were found to be differentially represented (P < 0.01) in at least one pairwise comparison (Table 1). Some transcripts found to increase in representation from the MPII to the 2-cell stage include protein kinases, PRKACA and CKS1, as well as the metabolism-related gene, PTTG1. These same transcripts were also found to decrease in representation from the 2-cell to the PCM stage. RPL15 (translation) and FTH1 (immune function) were both more highly represented in the PCM than in the 2-cell stage. From PCM to IVTBL, we saw an increase in RPS11, another translation-related transcript. When comparing blastocyst-stage embryos from different production techniques, several transcripts involved in energy production (e.g., COX7B and COX8A) were found to be more highly represented in the NTBL than in the IVTBL. COX8A was also more highly represented in the IVVBL than in the IVTBL. By investigating these differentially represented transcripts, we will be able to better understand the developmental implications of embryo manipulation. We may also be able to better develop reproductive technologies that lead to in vitro- and nuclear transfer-derived embryos which more closely follow a normal program of development. Table 1. Differentially represented transcripts between developmental stages

scholarly journals Phenolic Profile of Herbal Infusion and Polyphenol-Rich Extract from Leaves of the Medicinal Plant Antirhea borbonica: Toxicity Assay Determination in Zebrafish Embryos and Larvae

Molecules ◽  
2020 ◽  
Vol 25 (19) ◽  
pp. 4482
Author(s):  
Bryan Veeren ◽  
Batoul Ghaddar ◽  
Matthieu Bringart ◽  
Shaymaa Khazaal ◽  
Marie-Paule Gonthier ◽  
...  
Keyword(s):  
Antioxidant Properties ◽  
Spinal Curvature ◽  
Therapeutic Effects ◽  
Zebrafish Embryos ◽  
Endemic Plant ◽  
Fish Embryo ◽  
Liquid Chromatography Tandem Mass ◽  
Pericardial Edema ◽  
Living Organisms

Antirhea borbonica (A. borbonica) is an endemic plant from the Mascarene archipelago in the Indian Ocean commonly used in traditional medicine for its health benefits. This study aims (1) at exploring polyphenols profiles from two types of extracts—aqueous (herbal infusion) and acetonic (polyphenol rich) extracts from A. borbonica leaves—and (2) at evaluating their potential toxicity in vivo for the first time. We first demonstrated that, whatever type of extraction is used, both extracts displayed significant antioxidant properties and acid phenolic and flavonoid contents. By using selective liquid chromatography–tandem mass spectrometry, we performed polyphenol identification and quantification. Among the 19 identified polyphenols, we reported that the main ones were caffeic acid derivatives and quercetin-3-O-rutinoside. Then, we performed a Fish Embryo Acute Toxicity test to assess the toxicity of both extracts following the Organisation for Economic Cooperation and Development (OECD) guidelines. In both zebrafish embryos and larvae, the polyphenols-rich extract obtained by acetonic extraction followed by evaporation and resuspension in water exhibits a higher toxic effect with a median lethal concentration (LC50: 5.6 g/L) compared to the aqueous extract (LC50: 20.3 g/L). Our data also reveal that at non-lethal concentrations of 2.3 and 7.2 g/L for the polyphenol-rich extract and herbal infusion, respectively, morphological malformations such as spinal curvature, pericardial edema, and developmental delay may occur. In conclusion, our study strongly suggests that the evaluation of the toxicity of medicinal plants should be systematically carried out and considered when studying therapeutic effects on living organisms.

59 Selected Reaction Monitoring-Based Absolute Quantification of Developmentally Relevant Proteins in Early Bovine Embryos Reveals Differences Between In Vitro and In Vivo Embryo Culture and Between Different Maternal Metabolic Stages

2018 ◽  
Vol 30 (1) ◽  
pp. 168
Author(s):  
G. J. Arnold ◽  
K. Gegenfurtner ◽  
T. Frohlich ◽  
D. R. Deutsch ◽  
P. Salvetti ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Developmental Process ◽  
Absolute Quantification ◽  
Early Embryogenesis ◽  
Selected Reaction Monitoring ◽  
Reaction Monitoring ◽  
Cell Stage ◽  
Cell Potential

Early embryogenesis is a highly complex developmental process, accompanied by a plethora of changes at the morphological and molecular level. Particularly at the level of proteins, these changes are still poorly characterised and understood. During the first cleavages, the embryo depends mainly on maternal transcripts and proteins that were accumulated and stored during oogenesis until embryonic genome activation (EGA) occurs. In the bovine system, the major EGA takes place at the 8- to 16-cell stage. However, we recently demonstrated by liquid chormatography-tandem mass spectrometry (LC-MS/MS)-based holistic proteome approaches that despite transcriptional and translational silencing, the proteome of the early embryo is highly dynamic (Deutsch et al. 2014; Demant et al. 2015). Based on these findings, we established a targeted LC-MS/MS approach based on multiplexed selected reaction monitoring (mSRM), which facilitates an absolute quantification of 27 proteins relevant in early embryogenesis. Each protein is targeted by 2 independent peptides to facilitate highly reliable quantifications. Nine characteristic developmental stages from germinal vesicle oocyte to hatched blastocyst were analysed (n = 6 per stage), and absolute protein contents are reported as femtomole per embryo, with limits of quantification (LOQ) down to 100 attomoles per embryo. Based on their abundance profiles during maturation, zygote formation, and embryonic development, the 27 proteins could be grouped into 6 SOTA clusters. By principal component analysis (PCA), absolute SRM quantifications of only 9 selected proteins were shown to discriminate between all 9 developmental stages analysed, thus providing molecular fingerprints significant for each developmental stage. We used the 27-plex SRM assay as a powerful readout tool and demonstrated substantial quantitative differences between embryos derived from a well-established in vitro culture system and embryos transferred into the oviduct of living animals for 2 days (in vivo culture). Furthermore, in vivo development of embryos in animals differing in their metabolic stress levels led to significant alterations in the 27-plex SRM profiles. This work was supported by a grant to GJA from Deutsche Forschungsgemeinschaft DFG FOR1041 ‘Germ Cell Potential’ AR 362/7-1 and European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement n° 312097 - FECUND.

scholarly journals Mutant MAPK7-Induced Idiopathic Scoliosis is Linked to Impaired Osteogenesis

2018 ◽  
Vol 48 (3) ◽  
pp. 880-890 ◽  
Author(s):  
Taifeng Zhou ◽  
Chong Chen ◽  
Caixia Xu ◽  
Hang Zhou ◽  
Bo Gao ◽  
...  
Keyword(s):  
Idiopathic Scoliosis ◽  
Western Blotting ◽  
Messenger Rna ◽  
Human Mesenchymal Stem Cells ◽  
Control Group ◽  
Zebrafish Embryos ◽  
Small Interfering Rnas ◽  
Cell Stage ◽  
Alizarin Red ◽  
The One

Background/Aims: Three rare MAPK7 variants that predispose individuals to adolescent idiopathic scoliosis have previously been identified. However, the mechanism underlying the effects of the mutations remain unknown. Methods: Human mesenchymal stem cells (hMSCs) were isolated from both patients and healthy volunteer donors, and MAPK7 expression was detected by western blotting and real-time quantitative PCR (RT-qPCR). Zebrafish embryos were injected with mapk7 morpholinos or co-injected with morpholinos and wild-type (WT) MAPK7 messenger RNA (mRNA) at the one-cell stage, followed by calcein staining to evaluate bone formation. hMSCs were transfected with MAPK7 small interfering RNAs and osteogenesis was induced for 14 days. Alizarin red staining was performed and osteoblast markers were detected by western blotting and RT-qPCR. Since RPS6KA3 is a downstream target of MAPK7 and plays an important role in the osteogenesis, zebrafish embryos were then injected with rps6ka3 morpholinos, or co-injected with rps6ka3 or mapk7 morpholinos and WT RPS6KA3 mRNA at the one-cell stage. Results: MAPK7 expression in the patient group was much lower than in the control group. Morpholino-induced mapk7 knockdown in zebrafish embryos led to body curvature, which was significantly reversed by WT MAPK7 mRNA. Calcein staining revealed that mapk7-knockdown delayed the ossification of the vertebrae. MAPK7 silencing in hMSCs impaired osteogenesis and downregulated osteoblast marker expression. Morpholino-induced rps6ka3-knockdown in zebrafish embryos led to body curvature, which was reversed by WT RPS6KA3 mRNA. Interestingly, RPS6KA3 mRNA also partially reversed the phenotype induced by mapk7 morpholinos. Conclusion: Impaired osteogenesis is linked to mutant MAPK7-induced idiopathic scoliosis , and RPS6KA3 may play an important role in this process.

scholarly journals Direct Visualization of Live Zebrafish Glycans via Single‐Step Metabolic Labeling with Fluorophore‐Tagged Nucleotide Sugars

2019 ◽  
Vol 58 (40) ◽  
pp. 14327-14333 ◽  
Author(s):  
Senlian Hong ◽  
Pankaj Sahai‐Hernandez ◽  
Digantkumar Gopaldas Chapla ◽  
Kelley W. Moremen ◽  
David Traver ◽  
...  
Keyword(s):  
Single Step ◽  
Metabolic Labeling ◽  
Direct Visualization ◽  
Nucleotide Sugars

Open-pulled straw (OPS) vitrification of in vitro fertilised mouse embryos at various stages

2008 ◽  
Vol 56 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Chang-Liang Yan ◽  
Qi-En Yang ◽  
Guang-Bin Zhou ◽  
Yun-Peng Hou ◽  
Xue-Ming Zhao ◽  
...  
Keyword(s):  
Survival Rate ◽  
Developmental Stages ◽  
Developmental Rate ◽  
Blastocyst Stage ◽  
Significant Loss ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Fresh Embryos

The present study was designed to investigate the cryotolerance of in vitro fertilised (IVF) mouse embryos at various preimplantation developmental stages. IVF mouse embryos were vitrified by the open-pulled straw (OPS) method. After warming, embryos were morphologically evaluated and assessed by their development to blastocysts, hatched blastocysts or term. The results showed that a high proportion (93.3–100.0%) of vitrified embryos at all developmental stages were morphologically normal after recovery. The developmental rate of vitrified 1-cell embryos to blastocyst (40.0%) or hatched blastocyst (32.7%) or term (9.3%) was significantly lower than that from other stages (P < 0.05). Vitrified embryos from 2-cell to early blastocyst stage showed similar blastocyst (71.8–89.5%) and hatched blastocyst rates (61.1–69.6%) and could develop to term without a significant loss of survival compared with those of fresh embryos (P > 0.05). Vitrified 2-cell embryos showed the highest survival rate in vivo (50.6%, 88/174), compared with that from other stages (9.3–30.5%, P < 0.05). The data demonstrate that the OPS method is suitable for the cryopreservation of IVF mouse embryos from 2-cell stage to early blastocyst stage without a significant loss of survival. Embryos at the 2-cell stage had the best tolerance for cryopreservation in the present study.

88 VARIABLE DNA METHYLATION PROFILES AT IMPRINTED LOCI IN BOVINE EARLY PRE-IMPLANTATION EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 192
Author(s):  
A. M. O'Doherty ◽  
D. Magee ◽  
M. E. Beltman ◽  
S. Mamo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Developmental Stages ◽  
Transcript Abundance ◽  
Bovine Embryo ◽  
Differentially Methylated Regions ◽  
Cell Stage ◽  
Imprinted Gene ◽  
Genomic Imprints ◽  
Time Point

The DNA methylation imprints, at maternally imprinted gene differentially methylated regions, are established during the postnatal growth stage of oogenesis, with paternal imprints being acquired in the perinatal prospermatagonia. Murine DNA methylation marks, at imprinted loci, are widely regarded to be resistant to post-fertilization demethylation events that occur in the paternal pronucleus of the zygote and to passive demethylation of the maternally derived genomic content from cleavage to the 16-cell stage. However, the DNA methylation profile of bovine imprinted genes following fertilization remains unknown. The objective of the current study was to analyze the methylation dynamics at several imprinted gene differentially methylated regions during bovine embryo development. In addition, a previously published RNA-seq database (Mamo et al. 2011 Biol. Reprod.) was mined for transcript abundance of genes associated with establishing and maintaining genomic imprints. Single in vivo blastocysts (Day 7), hatched ovoid embryos (Day 14), filamentous embryos (Day 17), and implanting conceptii (Day 25) were collected (n = 4–9, per time point) from beef heifers. Genomic DNA was isolated and bisulfite modified, using the EZ DNA methylation direct kit (Zymo, Irvine, CA, USA), and used as template in bisulfite PCR reactions. The PCR products were verified by agarose gel electrophoresis and subsequently pyrosequenced. Observed methylation values were most highly variable in Day 7 blastocysts, with values ranging between 13 and 44% (IGF2R), 5 and 63% (PEG10), 7 and 59% (MEST), 3 and 61% (SNRPN), 12 and 64% (PLAGL1), and 20 and 32% (H19). There was a marked reduction in variability as embryonic development progressed, with values at Day 25 ranging from 37 to 41% (IGF2R), 34 to 38% (PEG10), 31 to 37% (MEST), 36 to 40% (SNRPN), 17 to 26% (PLAGL1), and 25 to 30% (H19). Statistical analysis (Levene’s test for equal variance) of methylation values for each gene at each time point confirmed that the methylation values observed in Day 7 embryos were significantly variable (P < 0.05) when compared with later developmental stages. Concordant with this finding, RNA transcript levels of associated methylation machinery genes DNMT3A, DNMT3B, and TRIM28 progressively increased from Day 7 to 13 and subsequently decreased from Day 13 to 16. Taken together our results demonstrate that in cattle DNA methylation marks, at imprinted loci, are highly variable at the blastocyst stage and are progressively stabilized with increasing days post-fertilization. This stabilization of imprint is coordinated with a window of increased levels of associated methylation machinery transcripts. Work presented here provides evidence of a novel mechanism for bovine embryonic DNA methylation imprint maintenance. This work was funded by SFI grant number 07/SRC/B1156.

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