132 Abundance and activity of metabolic enzymes in bovine cumulus cells derived from ovarian samples under variable physiological conditions

2022 ◽  
Vol 34 (2) ◽  
pp. 304
Author(s):  
S. Gebremedhn ◽  
M. Tannous ◽  
E. Natera ◽  
B. Krueger ◽  
M. Ambrogi ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Metabolic Enzymes ◽  
Physiological Conditions

133 The activity of metabolic enzymes in bovine oocytes derived from ovaries with heterogenous physiological conditions

2022 ◽  
Vol 34 (2) ◽  
pp. 304
Author(s):  
S. Gebremedhn ◽  
M. Ambrogi ◽  
B. Krueger ◽  
E. Natera ◽  
M. Tannous ◽  
...  
Keyword(s):  
Metabolic Enzymes ◽  
Physiological Conditions ◽  
Bovine Oocytes

127 Temporal regulation of molecular pathways, metabolic enzymes and growth factor receptors during bovine oocyte maturation in vitro

2019 ◽  
Vol 31 (1) ◽  
pp. 189
Author(s):  
S. Rajput ◽  
J. Becker ◽  
Y. Yuan ◽  
W. Schoolcraft ◽  
R. Krisher
Keyword(s):  
Growth Factor ◽  
Oocyte Maturation ◽  
Growth Factor Receptors ◽  
Cumulus Cells ◽  
Metabolic Enzymes ◽  
Molecular Pathways ◽  
Temporal Regulation ◽  
Maturation In Vitro ◽  
Significant Change

Although great efforts have been made to improve in vitro oocyte maturation (IVM) medium, we have yet to achieve competence equivalent to in vivo-matured oocytes. The failure in development of culture conditions for IVM yielding high quality eggs is attributed to an incomplete understanding of molecular pathways regulating oocyte and cumulus cell metabolism. The objective of the present study was to characterise the expression and functional activity of cell signalling pathways (mTOR, AKT, 4EBP1, ERK1/2), metabolic enzymes (PKM2, PDH, LDHA, AMPK), and growth factor receptors (IGF1R, IGFIIR, EGFR, FGFR1) in bovine oocytes and cumulus cells before and after in vitro maturation. In vitro-derived cumulus-oocyte complexes were collected at germinal vesicle (GV) and metaphase II (MII) stages (20 cumulus-oocyte complexes per stage; n=3 replicates) and subjected separately to Western blot analysis using antibodies against both phosphorylated (p) and total (t) protein abundance; the ratio p:t was used to determine the activity of each pathway. Results demonstrate increased (P<0.05) mTOR and ERK1/2 signalling, with no change in AKT and 4EBP1 activity, in oocytes during IVM. We observed increased (P<0.05) abundance of oocyte t-ERK from the GV to MII stage, but total expression of AKT, mTOR and 4EBP1 did not change. In cumulus cells, there was a significant (P<0.05) reduction in mTOR and 4EBP1, an increase in AKT, and no significant change in ERK activity. Analysis of metabolic enzymes in oocytes demonstrated increased (P<0.05) PDH, reduced AMPK, and unchanged PKM2 and LDHA phosphorylation during IVM. However, increased expression of t-PKM2 abundance was observed from the GV to MII stage. In cumulus cells, tAMPK abundance was reduced (P>0.05), but no significant change was observed in the activity of other metabolic enzymes analysed during IVM. Finally, we observed abundant expression of IGF2R in the oocyte compared with other growth factor receptors analysed, although IGF2R was significantly (P<0.05) reduced from GV to MII oocytes. In cumulus cells, both IGF1R and IGF2R were highly abundant compared with EGFR and FGFR but did not change during IVM. Data were analysed using one-way ANOVA. Results suggest that regulatory mechanisms including AKT/mTOR/4EBP1 and ERK are entirely different in oocytes and cumulus cells during maturation. An increase in the inhibitory phosphorylation of oocyte PDH (S293) toward the end of maturation suggests low metabolism of pyruvate via the Krebs cycle at that time. Similarly, dephosphorylation of AMPK (T172) suggests reduced AMPK activity and reduced fatty acid oxidation in mature oocytes. In addition, temporal regulation of IGF1R in the oocyte and EGFR in cumulus cells suggests an important role for these growth factor receptors during maturation and that these growth factors could be used to improve IVM medium in the bovine. Collectively, these results increase our understanding of the molecular pathways regulating oocyte metabolism during maturation and provide a strategy to improve the IVM environment for assisted reproductive technology.

scholarly journals Phosphoproteome Study of Escherichia coli Devoid of Ser/Thr Kinase YeaG During the Metabolic Shift From Glucose to Malate

2021 ◽  
Vol 12 ◽  
Author(s):  
Abida Sultan ◽  
Carsten Jers ◽  
Tariq A. Ganief ◽  
Lei Shi ◽  
Meriem Senissar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Isotope Labeling ◽  
Isocitrate Lyase ◽  
Metabolic Enzymes ◽  
Metabolic Phenotype ◽  
Lag Phase ◽  
Metabolic Shift ◽  
Physiological Conditions ◽  
Starting Point

Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.

Ultrastructure of Giant Mitochondria and Their Inclusions in Schwann Cells of Bovine Splenic Nerve

1974 ◽  
Vol 32 ◽  
pp. 194-195
Author(s):  
Å. Thureson-Klein
Keyword(s):  
Schwann Cells ◽  
Cell Organelles ◽  
Giant Mitochondria ◽  
Matrix Density ◽  
Physiological Conditions ◽  
Unmyelinated Axons ◽  
Internal Structures ◽  
Structural Abnormalities ◽  
Cytotoxic Compounds ◽  
Cell Components

Giant mitochondria of various shapes and with different internal structures and matrix density have been observed in a great number of tissues including nerves. In most instances, the presence of giant mitochondria has been associated with a known disease or with abnormal physiological conditions such as anoxia or exposure to cytotoxic compounds. In these cases degenerative changes occurred in other cell organelles and, therefore the giant mitochondria also were believed to be induced structural abnormalities.Schwann cells ensheating unmyelinated axons of bovine splenic nerve regularly contain giant mitochondria in addition to the conventional smaller type (Fig. 1). These nerves come from healthy inspected animals presumed not to have been exposed to noxious agents. As there are no drastic changes in the small mitochondria and because other cell components also appear reasonably well preserved, it is believed that the giant mitochondria are normally present jin vivo and have not formed as a post-mortem artifact.

Immunocytochemical detection of hepatic carbohydrate metabolic enzymes: Improved resolution with polyethylene glycol embedding and visio-bond semithin sections

1992 ◽  
Vol 50 (1) ◽  
pp. 792-793
Author(s):  
Kuixiong Gao ◽  
Randal E. Morris ◽  
Bruce F. Giffin ◽  
Robert R. Cardell
Keyword(s):  
Polyethylene Glycol ◽  
Glycogen Phosphorylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthase ◽  
Metabolic Enzymes ◽  
Silver Stain ◽  
Accurate Localization ◽  
Immunocytochemical Detection ◽  
Semithin Sections ◽  
Parenchymal Cells

Several enzymes are involved in the regulation of anabolic and catabolic pathways of carbohydrate metabolism in liver parenchymal cells. The lobular distribution of glycogen synthase (GS), phosphoenolpyruvate carboxykinase (PEPCK) and glycogen phosphorylase (GP) was studied by immunocytochemistry using cryosections of normal fed and fasted rat liver. Since sections of tissue embedded in polyethylene glycol (PEG) show good morphological preservation and increased detectability for immunocytochemical localization of antigenic sites, and semithin sections of Visio-Bond (VB) embedded tissue provide higher resolution of cellular structure, we applied these techniques and immunogold-silver stain (IGSS) for a more accurate localization of hepatic carbohydrate metabolic enzymes.

Freeze-fracture observations on changes in the distribution of Filipin-sterol complexes during epididymal maturation and capacitation of boar spermatozoa

1990 ◽  
Vol 48 (3) ◽  
pp. 90-91
Author(s):  
N. Seki ◽  
Y. Toyama ◽  
T. Nagano
Keyword(s):  
Plasma Membrane ◽  
Essential Role ◽  
Freeze Fracture ◽  
Final Concentration ◽  
Freeze Fracturing ◽  
Physiological Conditions ◽  
Epididymal Maturation ◽  
Cacodylate Buffer ◽  
Sperm Membrane ◽  
Boar Spermatozoa

It is believed that i ntramembra.nous sterols play an essential role in membrane stability and permeability. To investigate the distribution changes of sterols in sperm membrane during epididymal maturation and capacitation, filipin has been used as a cytochemical probe for the detection for membrane sterols. Using this technique in combination with freeze fracturing, we examined the boar spermatozoa under various physiological conditions.The spermatozoa were collected from: 1) caput, corpus and cauda epididymides, 2) sperm rich fraction of ejaculates, and 3)the uterus 2hr after natural coition. They were fixed with 2.5% glutaraldehyde in 0.05M cacodylate buffer (pH 7.4), and treated with the filipin solution (final concentration : 0.02.0.05%) for 24hr at 4°C with constant agitation. After the filipin treatment, replicas were made by conventional freeze-fracture technique. The density of filipin-sterol complexes (FSCs) was determined in the E face of the plasma membrane of head regions.

Influence of stable iodine on the uptake of the thyroid – model vs. experiment

2001 ◽  
Vol 40 (01) ◽  
pp. 31-37 ◽  
Author(s):  
U. Wellner ◽  
E. Voth ◽  
H. Schicha ◽  
K. Weber
Keyword(s):  
Time Course ◽  
Compartment Model ◽  
The Body ◽  
Iodine Uptake ◽  
Model Calculations ◽  
Physiological Conditions ◽  
Iodine Supply ◽  
Predicted Values ◽  
The Individual ◽  
Stable Iodine

Summary Aim: The influence of physiological and pharmacological amounts of iodine on the uptake of radioiodine in the thyroid was examined in a 4-compartment model. This model allows equations to be derived describing the distribution of tracer iodine as a function of time. The aim of the study was to compare the predictions of the model with experimental data. Methods: Five euthyroid persons received stable iodine (200 μg, 10 mg). 1-123-uptake into the thyroid was measured with the Nal (Tl)-detector of a body counter under physiological conditions and after application of each dose of additional iodine. Actual measurements and predicted values were compared, taking into account the individual iodine supply as estimated from the thyroid uptake under physiological conditions and data from the literature. Results: Thyroid iodine uptake decreased from 80% under physiological conditions to 50% in individuals with very low iodine supply (15 μg/d) (n = 2). The uptake calculated from the model was 36%. Iodine uptake into the thyroid did not decrease in individuals with typical iodine supply, i.e. for Cologne 65-85 μg/d (n = 3). After application of 10 mg of stable iodine, uptake into the thyroid decreased in all individuals to about 5%, in accordance with the model calculations. Conclusion: Comparison of theoretical predictions with the measured values demonstrated that the model tested is well suited for describing the time course of iodine distribution and uptake within the body. It can now be used to study aspects of iodine metabolism relevant to the pharmacological administration of iodine which cannot be investigated experimentally in humans for ethical and technical reasons.

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

1993 ◽  
Vol 70 (05) ◽  
pp. 867-872 ◽  
Author(s):  
Dingeman C Rijken ◽  
Gerard A W de Munk ◽  
Annie F H Jie
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Single Chain ◽  
Physiological Conditions ◽  
Amino Terminal ◽  
Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

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