374 SEX-DEPENDENT METABOLIC DIFFERENCES OF BOVINE PREIMPLANTATION EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 343
Author(s):  
R. G. Sturmey ◽  
P. Bermejo-Alvarez ◽  
A. Gutierrez-Adan ◽  
D. Rizos ◽  
H. J. Leese ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cellular Damage ◽  
Preimplantation Embryos ◽  
Fluid Medium ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Metabolic Marker

Sex-dependent differences in mammalian embryo phenotype are apparent at the preimplantation stage of development, before the appearance of sex-specific cells. The ratio of male:female embryos may be modified by environmental factors such as maternal diet in vivo and the composition of embryo culture media in vitro. We have used amino acid profiling (AAP), a defined, non-invasive metabolic marker of developmental potential to compare the effect of sex on the metabolism of bovine preimplantation blastocysts and expanded blastocysts conceived in vivo (n = 35) or produced in vitro (n = 172). Blastocysts were incubated individually for 24 h in synthetic oviduct fluid medium plus a close-to-physiological mixture of amino acids. The depletion or appearance of 18 amino acids was measured using high-performance liquid chromatography. Blastocysts were then sexed by PCR and the outcome related to AAP. Amino acid depletion by in vitro-produced blastocysts was higher than in embryos conceived in vivo (P = 0.02). Net appearance of amino acids was higher in the medium from early blastocysts produced in vitro (P = 0.018) although this rise was lost at the expanded stage. There were marked differences in the amino acid profiles of male and female embryos produced in vitro: female embryos exhibited significantly increased depletion of arginine, glutamate, and methionine and appearance of glycine, while male embryos displayed increased depletion of phenylalanine, tyrosine, and valine. Overall, in vitro-produced blastocysts exhibited gender-specific differences in metabolic profiles of 7 out of 18 amino acids; in vivo-produced blastocysts exhibited differences in 2 out of 18 amino acids. These differences had disappeared by the expanded blastocyst stage. Our experiments reveal striking differences in the metabolism of preimplantation embryos conceived in vivo and in vitro, some of which, particularly in the case of the in vitro-produced embryos, are dependent on embryo sex. Moreover, in vivo-derived embryos tend to have a reduced metabolism consistent with the Quiet Embryo Hypothesis, which proposes that higher quality embryos have less molecular and cellular damage than those of a lower quality and thus have a reduced need to take up nutrients for repair processes. Supported by a Wellcome-VIP/University of York Fellowship to RGS.

scholarly journals Amino acid depletion and appearance during porcine preimplantation embryo development in vitro

Reproduction ◽  
2005 ◽  
Vol 130 (5) ◽  
pp. 655-668 ◽  
Author(s):  
Paul J Booth ◽  
Peter G Humpherson ◽  
Terry J Watson ◽  
Henry J Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Preimplantation Embryos ◽  
Type I ◽  
Preimplantation Embryo Development ◽  
Type Iv ◽  
Stage Of Development ◽  
Amino Acid Depletion

Preimplantation embryos can consume and produce amino acids in a manner dependent upon the stage of development that may be predictive of subsequent viability. In order to examine these relationships in the pig, patterns of net depletion and appearance of amino acids byin vitroproduced porcine preimplantation embryos were examined. Cumulus oocyte complexes derived from slaughterhouse pre-pubertal pig ovaries were matured for 40 h in defined TCM-199 medium (containing PVA) before being fertilised (Day 0) with frozen-thawed semen in Tris–based medium. After 6 h, presumptive zygotes were denuded and cultured in groups of 20, in NCSU-23 medium modified to contain 0.1 mM glutamine plus a mixture of 19 amino acids (aa) at low concentrations (0.02–0.11 mM) (NCSU-23aa). Groups of 2–20 embryos were removed (dependent on stage) on Day 0 (1 cell), Day 1 (two- and four-cells), Day 4 (compact morulae) and Day 6 (blastocysts) and placed in 4 μl NCSU-23aafor 24 h. After incubation, the embryos were removed and the spent media was analysed by HPLC. The net rate of amino acid depletion or appearance varied according to amino acid (P< 0.001) and, apart from serine and histidine, stage of development (P< 0.014). Glycine, isoleucine, valine, phenylalanine, tryptophan, methionine, asparagine, lysine, glutamate and aspartate consistently appeared, whereas threonine, glutamine and arginine were consistently depleted. Five types of stage-dependent trends could be observed: Type I: amino acids having high rates of net appearance on Day 0 that reached a nadir on Day 1 or 4 but subsequently increased by Day 6 (glycine, glutamate); Type II: those that exhibited lower rates of net appearance on Days 0 and 6 compared with the intermediate Days 1 and 4 (isoleucine, valine, phenylalanine, methionine, arginine); Type III: amino acids which showed a continuous fall in net appearance (asparagine, aspartate); Type IV: those that exhibited a steady fall in net depletion from Day 0 to Day 6 (glutamine, threonine); Type V: those following no discernable trend. Analysis of further embryo types indicated that presumptive polyspermic embryos on Day 0 had increased (P< 0.05) net rates of leucine, isoleucine, valine and glutamate appearance, and reduced (P< 0.05) net rates of threonine and glutamine depletion compared with normally inseminated oocytes. These data suggest that the net rates of depletion and uptake of amino acids by pig embryos vary between a) amino acids, b) the day of embryo development and, c) the type of embryos present at a given stage of development. The results also suggested that the net depletion and appearance rates of amino acids by early pig embryos might be more similar to those of the human than those of the mouse and cow.

Consumption of amino acids by bovine preimplantation embryos

1996 ◽  
Vol 8 (6) ◽  
pp. 945 ◽  
Author(s):  
RJ Partridge ◽  
HJ Leese
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Amino Acid Requirement ◽  
Zygote Stage

Bovine embryos produced in vitro from the putative zygote stage to the blastocyst stage, and blastocysts freshly flushed from the uterus, were cultured in a physiological mixture of amino acids. Depletion of amino acids from the medium and, in a few cases, their appearance, was measured by high performance liquid chromatography. Amino acids were depleted at widely differing rates. The depletion of amino acids was higher when embryos at later developmental stages were cultured, implying an increase in amino acid requirement with development. Threonine was the only amino acid to be depleted at all stages of development; depletion increased from 0.18 +/- 0.07 pmol embryo-1 h-1 at the putative zygote stage to 1.96 +/- 0.49 pmol embryo-1 h-1 at the blastocyst stage. Glutamine was depleted at the putative zygote stage and the 4-cell stage (0.76 +/- 0.05 and 0.94 +/- 0.10 pmol embryo-1 h-1 respectively), but was not significantly depleted at the later stages. Alanine was the only amino acid that appeared consistently in the medium and its production increased progressively throughout development. Aspartate, glutamate, threonine and lysine were depleted significantly by blastocysts derived both in vitro and in vivo; the embryos in vivo also depleted arginine, phenylalanine, isoleucine and tyrosine. These results indicate that individual amino acids are depleted at different rates by bovine preimplantation embryos and suggest that amino acid requirements change during development.

Effect of Cycloheximide on In Vitro and In Vivo Protein Synthesis by Certain Tissues of Rats and Pigeons with Special Reference to Muscle

1973 ◽  
Vol 51 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Njanoor Narayanan ◽  
Jacob Eapen
Keyword(s):  
Amino Acids ◽  
Body Weight ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Amino Acid Incorporation ◽  
Contrast Administration ◽  
Acid Incorporation ◽  
Tissue Homogenates

The effect of cycloheximide in vitro and in vivo on the incorporation of labelled amino acids into protein by muscles, liver, kidneys, and brain of rats and pigeons was studied. In vitro incorporation of amino acids into protein by muscle microsomes, myofibrils, and myofibrillar ribosomes was not affected by cycloheximide. In contrast, administration of the antibiotic into intact animals at a concentration of 1 mg/kg body weight resulted in considerable inhibition of amino acid incorporation into protein by muscles, liver, kidneys, and brain. This inhibition was observed in all the subcellular fractions of these tissues during a period of 10–40 min after the administration of the precursor. Tissue homogenates derived from in vivo cycloheximide-treated animals did not show significant alteration in in vitro amino acid incorporation with the exception of brain, which showed a small but significant enhancement.

scholarly journals Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

2020 ◽  
Author(s):  
Vera A van der Weijden ◽  
Meret Schmidhauser ◽  
Mayuko Kurome ◽  
Johannes Knubben ◽  
Veronika L Flöter ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Early Stage ◽  
Developmental Competence ◽  
Molecular Signaling ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Molecular Signaling Pathways ◽  
Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

2021 ◽  
Author(s):  
Babu Sudhamalla ◽  
Anirban Roy ◽  
Soumen Barman ◽  
Jyotirmayee Padhan
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Unnatural Amino Acids ◽  
Protein Protein Interactions ◽  
Site Specific ◽  
Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilized oocytes

1998 ◽  
Vol 10 (3) ◽  
pp. 279 ◽  
Author(s):  
Y. G. Jung ◽  
T. Sakata ◽  
E. S. Lee ◽  
Y. Fukui
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Culture Medium ◽  
Acid Metabolism ◽  
Essential Amino Acids ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Oviduct Fluid

The uptake and synthesis of 19 amino acids by fresh or frozen–thawed bovine blastocysts produced by parthenogenesis (PT) or in vitro fertilization (IVF) were compared in the present study. Fresh blastocysts, 180 h after IVF or PT activation, and frozen–thawed blastocysts, 168 h old and cultured for 12 h post-thawing, were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA, respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2: M2). In Experiment 1, when fresh or frozen–thawed PT blastocysts were cultured in M1, the uptake of glutamate (in fresh only), aspartate and arginine, and the synthesis of glutamine and alanine were significantly enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine, glycine, arginine and alanine were significantly taken up. It was found that the glutamine concentrations was significantly higher (P < 0.001) in the culture medium drops containing embryos than in the drops without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the uptake of aspartate and synthesis of alanine were greater (P < 0.01) than those by IVF blastocysts. When M2 was used, a significant (P < 0.01) production of serine, asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured in M1, fresh blastocysts depleted more aspartate and glutamate, and produced more glutamine and alanine than frozen–thawed blastocysts. When cultured in M2, frozen–thawed blastocysts depleted more threonine (P < 0.01) than fresh blastocysts. These results indicate that the uptake and synthesis of amino acids were different in fresh or frozen–thawed bovine blastocysts derived from PT or IVF. These differences in amino acid metabolism may be related to the viability of the blastocysts.

Amino acid fluxes in rat thin limb segments of Henle’s loop during in vitro microperfusion

1999 ◽  
Vol 277 (2) ◽  
pp. F204-F210 ◽  
Author(s):  
Olga H. Brokl ◽  
William H. Dantzler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Apparent Movement ◽  
Transepithelial Transport ◽  
Acid Transport ◽  
Henle's Loop ◽  
Vasa Recta ◽  
The Difference

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes ofl-[14C]alanine and [14C]taurine in thin limbs of Henle’s loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle’s loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle’s loop may occur via a paracellular route.

Correlation between osmoregulation and cell volume regulation

1987 ◽  
Vol 252 (4) ◽  
pp. R768-R773
Author(s):  
M. A. Lang
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Osmotic Pressure ◽  
Cell Volume ◽  
Free Amino Acids ◽  
Volume Regulation ◽  
Free Amino ◽  
Cell Volume Regulation

The euryhaline crab, Callinectes sapidus, behaves both as an osmoregulator when equilibrated in salines in the range of 800 mosM and below and an osmoconformer when equilibrated in salines above 800 mosM. There exists a close correlation between osmoregulation seen in the whole animal in vivo and cell volume regulation studied in vitro. Hyperregulation of the hemolymph osmotic pressure and cell volume regulation both occurred in salines at approximately 800 mosM and below. During long-term equilibration of the crabs to a wide range of saline environments, the total concentration of hemolymph amino acids plus taurine remained below 3 mM. During the first 6 h after an acute osmotic stress to the whole animal, the hemolymph osmotic pressure and Na activity gradually decreased, whereas the free amino acids remained below 3 mM. As the hemolymph osmotic pressure decreased below approximately 850 mosM, the amino acid level began to increase to 17-25 mM. This change was primarily due to increases in glycine, proline, taurine, and alanine. The likely source of the increase in hemolymph free amino acids in vivo is the free amino acid loss from muscle cells observed during cell volume regulation in vitro.

Incorporation of C14-amino acids into protein of isolated diaphragms: effect of epinephrine and norepinephrine

1960 ◽  
Vol 198 (1) ◽  
pp. 54-56 ◽  
Author(s):  
Ira G. Wool
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glucose Concentration ◽  
Muscle Protein ◽  
Effective Concentration ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Minimal Effective Concentration

When diaphragms isolated from normal rats were incubated with a C14-amino acid the addition of epinephrine or norepinephrine decreased incorporation of C14 into muscle protein. The inhibition occurred whether epinephrine was added in vitro or administered in vivo. The minimal effective concentration of epinephrine in vitro was 0.1 µg/ml. When the glucose concentration in the medium was raised to 300 mg % or more the epinephrine induced inhibition of amino acid incorporation into muscle protein was no longer observed.

Early Hematopoietic Progenitors of Dkc1 Hypomorphic Mutant Mice Display Decreased Proliferation Rate and an Impaired Control of Serine/Arginine-Rich Splicing Factor 4 (Srsf4) Translation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2937-2937
Author(s):  
Antonio R Lucena-Araujo ◽  
Bárbara A Santana-Lemos ◽  
Carol H Thome ◽  
Germano A Ferreira ◽  
Davide Ruggero ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Proliferation Rate ◽  
Splicing Factor ◽  
Differentially Expressed Proteins ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

Abstract The X-linked form of dyskeratosis congenita (X-DC) is caused by mutations in DKC1, which encodes for dyskerin: a putative pseudouridine synthase that mediate the posttranscriptional modification of ribosomal RNA (rRNA) through the conversion of uridine (U) to pseudouridine (Ψ). Patients with X-DC display defects in the pseudouridylation of ribosomal RNA that leads to translational upregulation of IRES-containing mRNAs and affects the affinity of the ribosome for these mRNAs. Studies in vivo and in vitro suggest that the pseudouridylation of ribosomal RNA is the underlying mechanism responsible for the enhanced susceptibility to cancer in these patients. Ruggero et al. have previously reported (Ruggero et al. Science. 2003 Jan 10;299(5604):259-62) that hypomorphic Dkc1m (Dkc1m) mice present pancytopenia associated with hypocellularity of the bone marrow (BM) and increased susceptibility to cancer, therefore constituting a reliable model to study the effect of impaired ribogenesis on hematopoiesis and oncogenesis. However, the cellular and molecular mechanisms leading to BM failure in X-DC remain unknown. Here, we describe the in vivo analysis of the proliferation rate of hematopoietic progenitors in Dkc1m mice and compare the proteomic profile of hematopoietic progenitors between Dkc1m mice and wild-type (WT) controls. For in vivo proliferation assays, 1mg of bromodeoxyuridine (BrdU) was injected intraperitoneally, every 6 hours during 24 hours, in 16 mice (eight WT and eight Dkc1m), and BM cells were harvested by flushing bone cavity, followed by immunofluorescence staining of incorporated BrdU and flow cytometric analysis. No differences were detected in the number of lineage-negative (Lin−), Sca1-positive, c-kit-negative (LSK−) cells, multipotent precursors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and immature B (B lin) and erythroid (Eryt) cells between Dkc1m and WT mice. Nevertheless, the BrdU incorporation was lower in LSK cells and CMPs from Dkc1m mice (P<0.05), indicating a lower proliferation rate. Using in vitro stable isotope labelling of amino acids (SILAC) hematopoietic progenitor cells were cultured in complete medium containing 10% fetal bovine serum and cytokines (6 ng/ml mIL-3, 10 ng/ml mIL-6 and 100 ng/ml mSCF). Of note, SILAC is one of the most applied approaches for quantitative proteomics, which uses labeled amino acids contain atoms of different isotopes in cell culture. Briefly, one cell population is cultured in unlabeled medium (control), while a second population is grown in medium substituted with a heavy amino acid (usually arginine 13C and/or lysine 15N). After 2-3 weeks culture, murine hematopoietic progenitors were collected and equal amounts of cells from Dkc1m and WT mice (labeled or not with heavy amino acid) were mixed for protein extraction and analyses. Using this approach, we identified about 3,500 differentially expressed proteins; including proteins related to mRNA assembling and splicing, chromatin remodeling, apoptosis and cell cycle arrest. Interestingly, one of the most differentially expressed proteins between WT and Dkc1m mice (WT light /Dkc1m heavy ratio: 18-fold) was the Serine/arginine-rich splicing factor 4(Srsf4); a member of the splicing factor family (SRSF1, SRSF3 and SRSF4) frequently associated with alternative splicing of genes related to hematopoietic progenitor cell differentiation. Altogether, our preliminary results reveal defects in the transcription/translation of specific mRNAs in Dkc1m cells. Additionally, it is conceivable that the down-regulation of Srsf4 protein could be associated with the low proliferative rate in DKC1m mice and explain the impairment of hematopoiesis in X-DC patients. Disclosures No relevant conflicts of interest to declare.

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