30 OXYGEN DEPRIVATION DOES NOT FURTHER AUGMENT MITOCHONDRIAL MEMBRANE POTENTIAL IN PHARMACOLOGICALLY TREATED FIBROBLASTS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER

2017 ◽  
Vol 29 (1) ◽  
pp. 122
Author(s):  
B. R. Mordhorst ◽  
S. N. Bogue ◽  
K. D. Wells ◽  
J. A. Green ◽  
R. S. Prather
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mitochondrial Membrane ◽  
Somatic Cells ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Donor Cells

Somatic cells commonly used in nuclear transfer primarily utilise the tricarboxylic acid cycle and cellular respiration for energy production. Comparatively, the metabolism of somatic cells contrasts that of cells within early embryos, which predominantly use glycolysis and exhibit Warburg Effect (WE)-like characteristics. We hypothesised that fibroblast cells can become more blastomere-like if driven either pharmacologically or by oxygen constraint and could result in improved in vitro embryonic development after somatic cell nuclear transfer (SCNT). The pharmaceuticals used (PS48 and CPI-613) should decrease mitochondrial use of the tricarboxylic acid (TCA) cycle and promote the PI3K pathway, respectively. Furthermore, we hypothesised that oxygen constraint (1.3%) would hinder TCA cycle activity and promote glycolysis. The goal was to achieve a WE-like effect in donor cells before nuclear transfer (NT) by treating Day 35 porcine fetal fibroblasts with CPI-613 (100 µM), PS48 (10 µM), both drugs combined (MIX), or as controls (CON, 0 µM) for 7 days under stepwise oxygen constraint (OC; 1.3%) or under normal conditions (ON; 5%). Three biological replicates were collected and data were analysed for main effect of treatment via GLM procedure of SAS 9.4 (SAS Institute Inc., Cary, NC, USA). To determine if our treatments affected mitochondria respiratory capacity (thereby TCA cycle capability) within embryos, we measured mitochondrial membrane potential (Δψm) using JC-10, a biphasic cationic dye. Mitotracker green (MTG) was used to estimate mitochondrial quantity. The percentage of cells with low Δψm was increased (P = 0.02) with any CPI or MIX treatment (treatments ≥ 95%) compared with OC-PS48 and both control (ON and OC) treatments (treatments ≥ 77.4%), whereas ON-PS48 had an intermediate level (90.4%; error = 4.9%). Contrary to our prediction, MTG intensity was lower across all ON treatments compared with OC treatments (NO treatments ≤ 736 AU v. OC treatments ≥ 872 AU; error = 23 AU; P < 0.01). Regardless of oxygen level, controls and PS48 treatments yielded the highest percentages of viable cells (treatments ≥ 94%) and OC-CPI and NO-MIX the lowest (treatments ≤ 86%) with NO-CPI and OC-MIX being intermediate (treatments ≥ 90%; error = 3%; P < 0.01). Oxygen constraint did not promote a reduction in mitochondrial membrane potential in pharmacologically treated fibroblasts. Additionally, intensity of MTG was increased in fibroblasts cultured under oxygen constraint compared with those cultured in 5% oxygen. Our results warrant further investigation of the mitochondrial changes occurring with oxygen deprivation in donor-cells. Experiments are underway to determine if gene expression in cells treated pharmacologically and with oxygen constraint are augmented, and whether these treatments will result in better development after SCNT. This study was funded by Food for the 21 st Century and NIH R01HD080636.

200 REPROGRAMMING OF Oct-4 FOLLOWING EQUINE SOMATIC CELL NUCLEAR TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 198
Author(s):  
T. Xiang ◽  
S. Walker ◽  
K. Gregg ◽  
W. Zhou ◽  
V. Farrar ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Donor Cells

Oct-4, a POU domain-containing transcription factor encoded by Pou5f1, is selectively expressed in pre-implantation embryos and pluripotent stem cells, but not in somatic cells. Because of such a unique expression feature, Oct-4 can serve as a useful reprogramming indicator in somatic cell nuclear transfer (SCNT). Compared with data of Oct-4 expression in mouse and bovine cloned embryos, little is known about this gene in equine nuclear transfer. In the present study, we investigated Oct-4 expression in donor cells, oocytes, and SCNT embryos to evaluate reprogramming of equine somatic cells following nuclear transfer. Horse ovaries were obtained from a local slaughterhouse and the oocytes collected from the ovaries were matured in vitro in an M199-based medium (Galli et al. 2003 Nature 424, 635) for 24 h. Donor cells were derived from biopsy tissue samples of adult horses and cultured for 1 to 5 passages. Standard nuclear transfer procedures (Zhou et al. 2008 Mol. Reprod. Dev. 75, 744–758) were performed to produce cloned embryos derived from equine adult somatic cells. Cloned blastocysts were obtained after 7 days of in vitro culture of reconstructed embryos. Total RNA were extracted using Absolutely RNA Miniprep/Nanoprep kits (Stratagen, La Jolla, CA) from oocytes (n = 200), donor cells, and embryos (n = 5). DNase I treatment was included in the procedure to prevent DNA contamination. Semiquantitative RT-PCR was performed with optimized cycling parameters to analyze Oct-4, GDF9, and β-actin in equine donor cells, oocytes, and cloned blastocysts. The RT-PCR products were sequenced to verify identity of the genes tested. The relative expression abundance was calculated by normalizing the band intensity of Oct-4 to that of β-actin in each analysis. No transcript of Oct-4 was detected in equine somatic cells used as donor nuclei, consistent with its expression patterns in other animal species, whereas Oct-4 was abundantly expressed in equine SCNT blastocysts derived from the same donor cell line. Oct-4 transcripts were also detected in equine oocytes and whether any maternally inherited Oct-4 mRNA persisted up to the blastocyst stage was unclear in this study. We selected GDF9 to address this question; GDF9 was abundantly detected in equine oocytes, consistent with its expression pattern in mouse and bovine, but not detected in donor cells and cloned blastocysts, suggesting that the GDF9 mRNA from the oocyte was degraded at least by the blastocyst stage. The results from this study imply occurrence of Oct-4 reprogramming in equine SCNT blastocysts, and future analysis for more developmentally important genes is needed to better understand reprogramming at molecular levels in this species.

26 DNA METHYLATION PATTERNS ARE APPROPRIATELY ESTABLISHED IN THE SPERM OF BULLS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER AFTER PASSAGE THROUGH THE GERMLINE

2009 ◽  
Vol 21 (1) ◽  
pp. 113 ◽  
Author(s):  
C. Couldrey ◽  
M. P. Green ◽  
D. N. Wells ◽  
R. S. F. Lee
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cpg Island ◽  
Somatic Cells ◽  
Cpg Sites ◽  
Donor Cells ◽  
Genomic Regions ◽  
Methylation Patterns

Cloning of domestic animals by somatic cell nuclear transfer (SCNT) has permitted the rescue of valuable genetics and has the potential to allow rapid dissemination of desirable traits in production animals through the use of cloned sires. Whilst cloned animals may show developmental deviations and aberrant DNA methylation suggestive of incomplete nuclear reprogramming, it is widely accepted that their offspring are normal, as any aberrant epigenetic marks are believed to be corrected on passage of the genome through the germline. We assessed the extent of reprogramming by comparing DNA methylation patterns in sperm of SCNT bulls (n = 4) with sperm from bulls generated by AI (n = 5) and with the nuclear donor somatic cells (adult skin fibroblasts). The genomic regions examined were 3 repetitive sequences (satellites 1, 2, and alpha) and CpG islands in 5 genes [HAND1, LIT1, MASH2, IGF2, Dickkopf-1(DKK-1)]. Semen was collected from 16-month-old bulls and assessed for volume, sperm number, morphology, and motility. DNA was extracted from washed sperm and somatic donor cells, bisulfite-treated and processed for quantification of CpG methylation using the Sequenom MassArray system. Methylation levels at individual CpG sites/groups of CpGs were compared between sample groups using the t-test with pooled variances. No apparent difference was detected in semen characteristics between SCNT and AI bulls. Sperm DNA methylation levels were very low in single copy genes with the exception of the CpG island in IGF2, which has previously been shown to be completely methylated in sperm. At all genomic regions examined, each CpG site or CpG groups were methylated to different levels, and each region had a distinctive profile, which was almost invariant between individual sperm samples from either the SCNT or AI bulls. In all sites examined, there were no significant differences in methylation profiles between sperm from SCNT and AI bulls. In contrast, DNA methylation profiles were significantly different between SCNT bull sperm and the donor cells. The exception was the CpG island in MASH2, which was essentially unmethylated in both. For the 3 satellite sequences along with LIT1, HAND1, and to a lesser extent, the DKK-1 region, DNA was significantly less methylated in sperm than in the donor cells. Only IGF2 was significantly more methylated in SCNT and AI sperm than in the donor cells at 10/25 CpG sites (P < 0.02). The results indicate that gametes from SCNT bulls had different epigenotypes from the donor somatic cells. This is the first molecular evidence that donor cell genomes have been reprogrammed in these SCNT bulls and that after going through the germline had acquired DNA methylation profiles that were similar to AI-derived bulls. It also suggests that any epigenetic aberrations that SCNT bulls may harbor are unlikely to be passed on to their offspring through their gametes. Supported by FRST contract C10X0311.

23 REDUCTION OF MITOCHONDRIAL FUNCTION, PROLIFERATION, AND GENE EXPRESSION IN FIBROBLAST DONOR CELLS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER BY CPI-613 AND PS48

2016 ◽  
Vol 28 (2) ◽  
pp. 141
Author(s):  
B. R. Mordhorst ◽  
S. L. Murphy ◽  
L. D. Spate ◽  
R. M. Ross ◽  
K. D. Wells ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Somatic Cell ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Tricarboxylic Acid Cycle ◽  
Pi3k Pathway ◽  
Live Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Main Effect

The morphology (spherical and without cristae) and metabolism (lowly functional) of mitochondria in early embryos and other rapidly proliferating cells exhibit a Warburg effect (WE)-like metabolism. A hallmark of the WE is the predominate use of glycolysis for energy production as opposed to the tricarboxylic acid cycle used by differentiated cells. Additionally, increased signalling of the PI3K pathway is correlated with an increase in glucose metabolism within cancer cells and is consistent with the WE. PS48 stimulates the PI3K pathway, and CPI-613 inhibits pyruvate dehydrogenase. The goal was to achieve a WE-like effect in donor cells before NT. Day 35 porcine fetal fibroblasts were treated as controls (CON, 0 μM) or with CPI (25, 50, or 100 μM) or PS48 (1, 5, 10 μM) for 7 days. Cytometry data were processed using SUMMIT software and analysed via GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA); all variables were analysed for the main effect of drug concentration. Trypan blue cell viability measures were analysed using GLM. For each collection day (i.e. Day 3, 5, and 7), all variables were analysed for the main effect of treatment, duration of culture, and their interaction. All mRNA expression as measured via the ΔΔ-ct method by qPCR was analysed using CT values in GLM for the main effect of drug treatment. Total number of cells and live cells at 120 h was decreased (P ≤ 0.03) in all PS48 treatments compared with CON cells (total cells: CON = 8.95 × 106 v. PS48 treatments ≤6.98 × 106; live cells: CON = 8.39 × 106 v. PS48 treatments ≤6.50 × 106). While the percentage live cells in CPI and CON cells did not differ (P ≥ 0.09), 100 μM decreased the number of total cells and live cells from that of CON for every time point by ~50% (P ≤ 0.02), whereas the other CPI treatments 25 and 50 μM were intermediate. Expression of PDK2 was reduced with 10 μM PS48 treatment compared with CON, and 50 and 100 μM CPI treated cells (P < 0.001; PS48 10 μM: 0.335 v. ≤1.012 other treatments). The CPI 100 μM and 10 μM PS48 concentrations decreased PKM M1 variant expression compared with CON and 50 μM CPI cells (P < 0.001; CPI 100 μM and PS48 10 μM <0.44; CPI 50 μM and CON >0.68). To determine the mitochondrial membrane potential, JC-10 was used. The percentage of cells with high mitochondrial membrane potential decreased (P = 0.04) with PS48 treatment (PS48 treatments ≤19.6%, control = 25.6%). Treatment with CPI also decreased (P ≤ 0.01) membrane potential and the percentages of cells (high function: CPI treatments ≤12.7 v. 25.6% in control; low function: CPI treatments ≥80.3 v. 74.3%). Because PS48 or CPI decrease mitochondrial membrane potential and the abundance of PKM M1, the metabolism of these potential donor cells may be more blastomere like. Experiments are underway to determine whether cells treated with PS48 or CPI will result in better development after somatic cell NT. This study was funded by Food for the 21st Century and NIH R01HD080636.

Successful cloning of coyotes through interspecies somatic cell nuclear transfer using domestic dog oocytes

2013 ◽  
Vol 25 (8) ◽  
pp. 1142 ◽  
Author(s):  
Insung Hwang ◽  
Yeon Woo Jeong ◽  
Joung Joo Kim ◽  
Hyo Jeong Lee ◽  
Mina Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Canis Lupus ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fusion Rate ◽  
Domestic Dogs ◽  
Domestic Dog ◽  
Canis Lupus Familiaris ◽  
Cloning Efficiency ◽  
Donor Cells

Interspecies somatic cell nuclear transfer (iSCNT) is an emerging assisted reproductive technology (ART) for preserving Nature’s diversity. The scarcity of oocytes from some species makes utilisation of readily available oocytes inevitable. In the present study, we describe the successful cloning of coyotes (Canis latrans) through iSCNT using oocytes from domestic dogs (Canis lupus familiaris or dingo). Transfer of 320 interspecies-reconstructed embryos into 22 domestic dog recipients resulted in six pregnancies, from which eight viable offspring were delivered. Fusion rate and cloning efficiency during iSCNT cloning of coyotes were not significantly different from those observed during intraspecies cloning of domestic dogs. Using neonatal fibroblasts as donor cells significantly improved the cloning efficiency compared with cloning using adult fibroblast donor cells (P < 0.05). The use of domestic dog oocytes in the cloning of coyotes in the present study holds promise for cloning other endangered species in the Canidae family using similar techniques. However, there are still limitations of the iSCNT technology, as demonstrated by births of morphologically abnormal coyotes and the clones’ inheritance of maternal domestic dog mitochondrial DNA.

Using a nano-flare probe to detect RNA in live donor cells prior to somatic cell nuclear transfer

2015 ◽  
Vol 40 (1) ◽  
pp. 7-15
Author(s):  
Bo Fu ◽  
Liang Ren ◽  
Di Liu ◽  
Jian-zhang Ma ◽  
Tie-zhu An ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Live Donor ◽  
Donor Cells

scholarly journals Effects of inflammatory and anti-inflammatory environments on the macrophage mitochondrial function

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Dong Ji ◽  
Jian-yun Yin ◽  
Dan-feng Li ◽  
Chang-tai Zhu ◽  
Jian-ping Ye ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Citrate Synthase ◽  
Tca Cycle ◽  
Raw 264.7 Cells ◽  
Metabolic Adaptation ◽  
Isocitrate Dehydrogenase 2 ◽  
Anti Inflammatory

AbstractMitochondrial response to inflammation is crucial in the metabolic adaptation to infection. This study aimed to explore the mitochondrial response under inflammatory and anti-inflammatory environments, with a focus on the tricarboxylic acid (TCA) cycle. Expression levels of key TCA cycle enzymes and the autophagy-related protein light chain 3b (LC3b) were determined in raw 264.7 cells treated with lipopolysaccharide (LPS) and metformin (Met). Additionally, reactive oxygen species (ROS) levels and mitochondrial membrane potential were assessed using flow cytometry. Moreover, 8-week-old C57BL/6J mice were intraperitoneally injected with LPS and Met to assess the mitochondrial response in vivo. Upon LPS stimulation, the expression of key TCA enzymes, including citrate synthase, α-ketoglutarate dehydrogenase, and isocitrate dehydrogenase 2, and the mitochondrial membrane potential decreased, whereas the levels of LC3b and ROS increased. However, treatment with Met inhibited the reduction of LPS-induced enzyme levels as well as the elevation of LC3b and ROS levels. In conclusion, the mitochondrial TCA cycle is affected by the inflammatory environment, and the LPS-induced effects can be reversed by Met treatment.

scholarly journals Differential In Vivo Binding Dynamics of Somatic and Oocyte-specific Linker Histones in Oocytes and During ES Cell Nuclear Transfer

2005 ◽  
Vol 16 (8) ◽  
pp. 3887-3895 ◽  
Author(s):  
Matthias Becker ◽  
Antje Becker ◽  
Faiçal Miyara ◽  
Zhiming Han ◽  
Maki Kihara ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Somatic Cells ◽  
Linker Histone ◽  
Binding Properties ◽  
Paternal Genome ◽  
Linker Histones ◽  
In Vivo Binding

The embryonic genome is formed by fusion of a maternal and a paternal genome. To accommodate the resulting diploid genome in the fertilized oocyte dramatic global genome reorganizations must occur. The higher order structure of chromatin in vivo is critically dependent on architectural chromatin proteins, with the family of linker histone proteins among the most critical structural determinants. Although somatic cells contain numerous linker histone variants, only one, H1FOO, is present in mouse oocytes. Upon fertilization H1FOO rapidly populates the introduced paternal genome and replaces sperm-specific histone-like proteins. The same dynamic replacement occurs upon introduction of a nucleus during somatic cell nuclear transfer. To understand the molecular basis of this dynamic histone replacement process, we compared the localization and binding dynamics of somatic H1 and oocyte-specific H1FOO and identified the molecular determinants of binding to either oocyte or somatic chromatin in living cells. We find that although both histones associate readily with chromatin in nuclei of somatic cells, only H1FOO is capable of correct chromatin association in the germinal vesicle stage oocyte nuclei. This specificity is generated by the N-terminal and globular domains of H1FOO. Measurement of in vivo binding properties of the H1 variants suggest that H1FOO binds chromatin more tightly than somatic linker histones. We provide evidence that both the binding properties of linker histones as well as additional, active processes contribute to the replacement of somatic histones with H1FOO during nuclear transfer. These results provide the first mechanistic insights into the crucial step of linker histone replacement as it occurs during fertilization and somatic cell nuclear transfer.

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

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