68 TRANSCRIPTIONAL GENOME ACTIVATION IN CANINE EMBRYOS COLLECTED IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 146 ◽  
Author(s):  
S. Chastant-Maillard ◽  
C. Viaris de Lesegno ◽  
S. Thoumire ◽  
M. Chebrout ◽  
K. Reynaud
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Laser Scanning ◽  
Transcriptional Activity ◽  
Staining Intensity ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Stage ◽  
Morula Stage

Early embryonic stages are supported by maternal transcripts from the oocyte cytoplasm. Progressive transcription of embryonic genome is a key step for further embryonic development, especially during in vitro culture. To date, in vitro culture from fertilization to the blastocyst stage is inefficient in the canine species. The objective of this work was to identify minor and major activation in in vivo-produced dog embryos. Ovariectomies were performed in 31 Beagle bitches from 102 to 266 h after ovulation (post-ov), precisely timed by transabdominal ultrasonography. Embryos were collected by tubal flushing with M199-Hepes and immediately transferred into transcription buffer. Transcriptional activity was evaluated through 5-bromouridine 5′-triphosphate (BrUTP) incorporation in nascent RNA, without microinjection (Aoki et al. 1997). Oocytes from anoestrus ovaries were used as positive controls. 5-Bromouridine 5′-triphosphate incorporation was revealed by immunocytochemistry (anti-bromodeoxyuridine primary antibody) and embryonic DNA was stained by ethidium homodimer-2. Staining was quantified under laser scanning confocal microscopy. Transcriptional activity was calculated as (mean nuclear intensity – cytoplasmic mean intensity) × nuclear area and expressed in arbitrary units (AU). It was compared to 1 (similar intensity in nucleus and cytoplasm; i.e. no transcriptional activity) by t-test; levels of transcriptional activity were compared between stages by variance analysis. Seventy embryos (from 7 to 21 per stage) from 31 bitches were analysed, from 2 pronuclei to morula stage. Between 28 and 125 nuclei were quantified per stage. At each stage, transcriptional activity was calculated per embryo and per nucleus. A significant transcriptional activity was detected as early as the 2 pronuclei stage (102–132 h post-ov; 1.15 ± 0.05 AU). Transcriptional activity per embryo significantly increased between the 2- and the 4-cell stage and between the 8-cell and the morula stage. In early 8-cell embryos, staining intensity of the various nuclei was markedly heterogeneous within the same embryo, all nuclei being intensively stained from the late 8-cell stage onwards. Transcriptional activity per nucleus increased also from the 2- to the 4-cell stage (respectively, 120–161 h post-ov, 1.15 ± 0.02 AU and 133–154 h post-ov, 1.35 ± 0.04 AU) until the 8-cell stage (153–225 h post-ov, 5.12 ± 0.55 AU). Transcriptional levels at these 3 stages differed significantly. It decreased between the 8-cell and the morula stage (230–266 h post-ov, 3.06 ± 0.13 AU), probably reflecting the acquisition of a selectivity in gene expression at major activation, as in other species; Nothias et al. 1995). Addition of the transcriptional inhibitor α-amanitin during BrUTP incubation decreased the transcriptional activity by 60% (P < 0.05). Embryonic gene expression (minor activation) thus begins in the canine embryo as early as the 2 pronuclei stage, with major activation taking place during the 8-cell stage.

The Growth Inhibition of Targeted Pluronic/Formononetin Nanocomposite on Oral Squamous Cell Carcinoma In Vitro

2020 ◽  
Vol 12 (8) ◽  
pp. 1022-1029
Author(s):  
Ming Liu ◽  
Chen Lin ◽  
Xiaoqing Huang ◽  
Yuxiang Lin
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Growth Inhibition ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Laser Scanning ◽  
Critical Concentration ◽  
Laser Scanning Confocal Microscopy

Natural flavonoid formononetin (FN) has anticancer effects, but the hydrophobic structure, characteristics of the short half-life in vivo, limiting its clinical wide-ranging application. In this study, FN loaded Pluronic (PF)@folic acid (FA) micelles (FN-PF@FA), were prepared to improve the solubility, bioavailability and targeting. FA coupling PF was prepared by carbodiimide crosslinker chemical method, FN-PF@FA micelles were prepared by modified film hydration method, and compared the antitumor activity of FN loaded micelles with free FN In Vitro. The spherical smooth surface of FN-PF@FA micelles had smaller particle size (112.3±5.3 nm), high encapsulation efficiency (86.14±2.68%), high negative zeta potential (-25.8±0.57 mV), low critical concentration CMC (0.03 mg/mL), and better sustained release profile. In addition, FN-PF@FA micelles have a positive targeting effect on oral squamous cell carcinoma cells (SCC3). In 48 hours, the growth inhibition of 50% (GI50) was 28.6±1.2 μg/mL for FN and 17.4±0.78 μg/mL for FN-PF, the dose dropped by nearly 38.46%. In addition, the GI50 value of FN-PF@FA was 9.5±0.3 μg/mL, 66.43% lower than FN and 44.83% lower than FN-PF. Furthermore, the laser scanning confocal microscopy revealed that the conjugation of FA significantly improves the active targeting ability of micelles. FN-PF@FA micelles have the potential to target the release of anticancer drugs with higher bioavailability, further provides a new avenue for the application of traditional Chinese medicine extract in oral malignant tumor.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

2020 ◽  
Vol 177 (2) ◽  
pp. 483-493
Author(s):  
Li Wang ◽  
Wenli Xu ◽  
Qi Zhou ◽  
Bojin Xu ◽  
Yunlu Sheng ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Calcium Influx ◽  
Underlying Mechanism ◽  
Laser Scanning Confocal Microscopy ◽  
Thyroid Cell ◽  
Dependent Manner ◽  
Class Iii ◽  
Rat Thyroid

Abstract PCB118, a 2,3′,4,4′,5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0–48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. On the other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2 (DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

scholarly journals Quantitative Analysis of CBP- and P300-Induced Histone Acetylations In Vivo Using Native Chromatin

2003 ◽  
Vol 23 (21) ◽  
pp. 7611-7627 ◽  
Author(s):  
Kirk J. McManus ◽  
Michael J. Hendzel
Keyword(s):  
Substrate Specificity ◽  
Histone Deacetylases ◽  
Laser Scanning ◽  
Laser Scanning Confocal Microscopy ◽  
Resolving Power ◽  
Creb Binding Protein ◽  
Histone Tails ◽  
Nonhistone Proteins

ABSTRACT In vivo, histone tails are involved in numerous interactions, including those with DNA, adjacent histones, and other, nonhistone proteins. The amino termini are also the substrates for a number of enzymes, including histone acetyltransferases (HATs), histone deacetylases, and histone methyltransferases. Traditional biochemical approaches defining the substrate specificity profiles of HATs have been performed using purified histone tails, recombinant histones, or purified mononucleosomes as substrates. It is clear that the in vivo presentation of the substrate cannot be accurately represented by using these in vitro approaches. Because of the difficulty in translating in vitro results into in vivo situations, we developed a novel single-cell HAT assay that provides quantitative measurements of endogenous HAT activity. The HAT assay is performed under in vivo conditions by using the native chromatin structure as the physiological substrate. The assay combines the spatial resolving power of laser scanning confocal microscopy with simple statistical analyses to characterize CREB binding protein (CBP)- and P300-induced changes in global histone acetylation levels at specific lysine residues. Here we show that CBP and P300 exhibit unique substrate specificity profiles, consistent with the developmental and functional differences between the two HATs.

scholarly journals Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

2021 ◽  
Vol 12 ◽  
Author(s):  
Ping He ◽  
Shu Li ◽  
Shengtao Xu ◽  
Huacai Fan ◽  
Yongfen Wang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Laser Scanning Confocal Microscopy ◽  
Transformation System ◽  
Scanning Confocal Microscopy ◽  
Bacillus Spp ◽  
Green Fluorescent ◽  
Race 4

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
A. Gad ◽  
U. Besenfelder ◽  
V. Havlicek ◽  
M. Hölker ◽  
M. U. Cinar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
In Vitro Culture ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Control Group ◽  
Transcriptome Profile ◽  
Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 153-161
Author(s):  
Jacek A. Modliński
Keyword(s):  
Survival Rate ◽  
Cytochalasin B ◽  
Single Case ◽  
Cell Stage ◽  
Morula Stage ◽  
Homozygous Diploid ◽  
Haploid Embryos ◽  
Early Developmental

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos, which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72–80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.

scholarly journals Studying the Neurovascular Unit: An Improved Blood–Brain Barrier Model

2009 ◽  
Vol 29 (12) ◽  
pp. 1879-1884 ◽  
Author(s):  
Christoph M Zehendner ◽  
Heiko J Luhmann ◽  
Christoph RW Kuhlmann
Keyword(s):  
Blood Brain Barrier ◽  
Laser Scanning ◽  
Neurovascular Unit ◽  
Cell Types ◽  
Laser Scanning Confocal Microscopy ◽  
Brain Barrier ◽  
Scanning Confocal Microscopy ◽  
Blood Brain

The blood–brain barrier (BBB) closely interacts with the neuronal parenchyma in vivo. To replicate this interdependence in vitro, we established a murine coculture model composed of brain endothelial cell (BEC) monolayers with cortical organotypic slice cultures. The morphology of cell types, expression of tight junctions, formation of reactive oxygen species, caspase-3 activity in BECs, and alterations of electrical resistance under physiologic and pathophysiological conditions were investigated. This new BBB model allows the application of techniques such as laser scanning confocal microscopy, immunohistochemistry, fluorescent live cell imaging, and electrical cell substrate impedance sensing in real time for studying the dynamics of BBB function under defined conditions.

Comparative expression analysis of embryonic development-related genes at different stages of parthenogenetic and in vitro fertilized embryos in caprine

Zygote ◽  
2013 ◽  
Vol 23 (2) ◽  
pp. 198-204 ◽  
Author(s):  
Renu Singh ◽  
Kuldeep Kumar ◽  
R. Ranjan ◽  
Manish Kumar ◽  
T. Yasotha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Developmental Competence ◽  
Cell Stage ◽  
Developmental Abnormalities ◽  
Aberrant Gene Expression ◽  
Comparative Expression Analysis ◽  
Morula Stage ◽  
Aberrant Gene ◽  
Parthenogenetic Embryos

SummaryAberrant gene expression occurs in parthenogenetic embryos due to abnormal epigenetic modifications in the genome that probably diminish viability and enhance developmental abnormalities in these embryos. In the present study, five developmentally important genes (HPRT1, Cx43, Sox2, Mest and IGF2R) were analysed at different stages in parthenotes (haploid and diploid) and compared with similar stages in in vitro fertilized (IVF) embryos. The results indicated that in haploid parthenotes expression of HPRT1 was upregulated (P < 0.05) only at the 2–4-cell stage whereas Cx43 expression was significantly (P < 0.05) downregulated in all stages as compared with the control. However, expression of this gene was upregulated (P < 0.05) in 2–4-cell and morula stages of diploid parthenotes. Expression of Sox2 was significantly (P < 0.05) downregulated in morula stage haploid parthenotes, whereas it was upregulated (P < 0.05) in 8–16-cell stage diploid embryos. The expression of Mest was upregulated (P < 0.05) at the 2–4-cell stage of both haploid and diploid parthenotes, whereas it was downregulated in 8–16-cell stage diploid embryos as compared with control. IGF2R expression was upregulated (P < 0.05) only in morula stage haploid and diploid parthenote as compared with control. These results indicate that parthenogenetic embryos showed aberrant gene expression of developmentally important genes such as HPRT1, Cx43, Sox2, Mest and IGF2R in comparison with IVF embryos, this finding may be one of the major reasons for the poor developmental competence of parthenogenetic embryos.

260 COMPARISON OF HOUSEKEEPING GENE EXPRESSION IN INDIVIDUAL 8-CELL STAGE MOUSE EMBRYOS PRODUCED UNDER DIFFERENT CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 246
Author(s):  
A. Baji Gal ◽  
S. Mamo ◽  
S. Bodo ◽  
A. Dinnyes
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Standard Error ◽  
Real Time Pcr ◽  
Housekeeping Gene ◽  
Mouse Embryos ◽  
Cell Stage ◽  
The Mean

Real-time PCR has the potential to accurately quantify the mRNA level of selected genes in single cells and individual pre-implantation-stage embryos. The goal of our study was to examine the variations in gene expression within individual embryos of the same stage and between embryos of the same stage but from different sources. In our study, we determined expression level of the 7 most commonly used housekeeping genes in 8-cell-stage mouse embryos produced under different culture conditions. Messenger RNA of 6 embryos each that was derived in vivo, or cultured in vitro from the zygote stage, or derived from oocytes activated parthenogenetically and developed in vitro were extracted individually followed by reverse transcription into cDNA. Optimized real-time PCR was performed for cytoplasmic beta-actin (Actb), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), H2A histone family, member Z (H2afz), hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1), ubiquitin C (Ubc), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and eukaryotic translation elongation factor 1 epsilon 1 (Eef1e1) genes. The results were analyzed, and the percentage standard error of the mean relative expression value was compared for all genes. All 7 genes were presented above the detection limit in all samples. One or two individual embryos showed 2- to 4-fold higher mRNA levels than the average for all genes in the group. The embryos cultured in vitro showed much higher expression levels of H2afz, Ppia, and Eef1e1 genes than those in the in vivo group. The parthenogenetic group was similar to the in vivo group in expression of Actb, H2afz, Hprt, and Eef1e1 genes, but showed significant differences (P &lt; 0.05; Student's t-test) compared to the in vitro group (Table 1). The percent standard error of the mean decreased gradually as the number of samples was increased. The 6 individual embryos in similar groups showed relatively low variability compared to embryos at similar stage but produced in different conditions. Interestingly, the parthenogenetic embryos showed a level of gene expression comparable to that of the in vivo ones, notwithstanding their culture in vitro. In conclusion, morphological observations and similarity in developmental stage alone cannot guarantee the uniformity of embryo samples, and a minimum of 4–6 replicates per treatment is needed. Moreover, we showed that culture condition itself has an effect on housekeeping gene expression, which, if neglected, might result in misinterpretation of data. Table 1.Relative expression values of the different culture groups (mean ±SE; n =6 embryos) This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No. 070246), and Hungarian National Science Fund (OTKA) (Grant No. T046171).

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