scholarly journals Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity

Reproduction ◽  
2008 ◽  
Vol 135 (2) ◽  
pp. 197-212 ◽  
Author(s):  
Helmut Torner ◽  
Nasser Ghanem ◽  
Christina Ambros ◽  
Michael Hölker ◽  
Wolfgang Tomek ◽  
...  
Keyword(s):  
Map Kinases ◽  
Protein Biosynthesis ◽  
Expression Profiles ◽  
Atp Synthesis ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Functional Markers ◽  
G6pdh Activity ◽  
Developmental Potential ◽  
Glucose 6 Phosphate Dehydrogenase

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here, we aim to identify molecular and functional markers associated with oocyte developmental potential when selected based on G6PDH activity. Immature compact cumulus–oocyte complexes were stained with brilliant cresyl blue (BCB) for 90 min. Based on their colouration, oocytes were divided into BCB−(colourless cytoplasm, high G6PDH activity) and BCB+(coloured cytoplasm, low G6PDH activity). The chromatin configuration of the nucleus and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement. The abundance and phosphorylation pattern of protein kinases Akt and MAP were estimated by Western blot analysis. A bovine cDNA microarray was used to analyse the gene expression profiles of BCB+and BCB−oocytes. Consequently, marked differences were found in blastocyst rate at day 8 between BCB+(33.1±3.1%) and BCB−(12.1±1.5%) oocytes. Moreover, BCB+oocytes were found to show higher phosphorylation levels of Akt and MAP kinases and are enriched with genes regulating transcription (SMARCA5), cell cycle (nuclear autoantigenic sperm protein,NASP) and protein biosynthesis (RPS274Aand mRNA for elongation factor 1α,EF1A). BCB−oocytes, which revealed higher mitochondrial activity and still nucleoli in their germinal vesicles, were enriched with genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10) and growth factor activity (bone morphogenetic protein 15,BMP15). This study has evidenced molecular and subcellular organisational differences of oocytes with different G6PDH activity.

254 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF OOCYTES SCREENED FOR THEIR DEVELOPMENTAL COMPETENCE BASED ON G6PDH ACTIVITY

2008 ◽  
Vol 20 (1) ◽  
pp. 207
Author(s):  
H. Torner ◽  
N. Ghanem ◽  
C. Ambros ◽  
M. Hoelker ◽  
W. Tomek ◽  
...  
Keyword(s):  
Map Kinase ◽  
Protein Biosynthesis ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Atp Synthesis ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
G6pdh Activity ◽  
Cresyl Blue

Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes (Alm 2005 Theriogenology 63, 2194–2205). However, the intrinsic molecular and subcellular characteristics of these oocytes have not yet been investigated. Here we aim to compare the developmental, molecular, and subcellular characteristics of oocytes selected using brilliant cresyl blue (BCB) staining based on G6PDH activity. Immature compact cumulus–oocyte complexes (COCs) were stained with 26 µm BCB (B-5388, Sigma-Alderich, Taufenkirchen, Germany) for 90 min. Based on their coloration, oocytes were divided into BCB– (colorless cytoplasm, high G6PDH activity) and BCB+ (colored cytoplasm, low G6PDH activity). The chromatin configuration and the mitochondrial activity of oocytes were determined by fluorescence labelling and photometric measurement (n = 337). The abundance and phosphorylation pattern of protein kinases Akt and MAP kinase were estimated by western blot analysis (n = 500). A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of BCB+ and BCB– oocytes (n = 580). BCB+ oocytes were found to result in a higher blastocyst rate (33.1 � 3.1%) until Day 8 of in vitro culture compared to BCB– ones (12.1 � 1.5%). Moreover, BCB+ oocytes showed higher phosphorylation levels of Akt and MAP kinase compared to the BCB– oocytes. After array data analysis, BCB+ oocytes were found to be enriched with genes regulating transcription (SMARCA5), cell cycle (NASP), and protein biosynthesis (RPS274A and EEF1A1), while the BCB– oocytes had a higher level of genes involved in ATP synthesis (ATP5A1), mitochondrial electron transport (FL405), calcium ion binding (S100A10), and growth factor activity (BMP-15). Independent real-time quantitative PCR validated 90% (9/10) of the genes investigated to be in agreement with the array expression profile. The study has shown evidence of differences in molecular and subcellular organization of oocytes with different G6PDH activity.

Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities

2010 ◽  
Vol 22 (5) ◽  
pp. 830 ◽  
Author(s):  
István Egerszegi ◽  
Hannelore Alm ◽  
József Rátky ◽  
Bassiouni Heleil ◽  
Klaus-Peter Brüssow ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Mitochondrial Activity ◽  
G6pdh Activity ◽  
Cresyl Blue ◽  
Meiotic Progression ◽  
Germinal Vesicles ◽  
Nuclear Development ◽  
Chromatin Configuration ◽  
Glucose 6 Phosphate Dehydrogenase

The aim of the present study was to investigate the developmental competence, mitochondrial characteristics and chromatin status of immature follicular porcine oocytes selected for their glucose-6-phosphate dehydrogenase (G6PDH) activity by brilliant cresyl blue (BCB) staining. In Experiment 1, the oocyte parameters were determined in parallel right after BCB staining (T0), after 22 h of in vitro maturation (IVM) (T22) and after 44 h of IVM (T44) (n = 496). BCB-stained oocytes (BCB+) at T0 were characterised by fibrillated chromatin filaments in their germinal vesicles (GV) and diakinesis stages whereas unstained (BCB–) oocytes at T0 contained in their GV mainly condensed stages of chromatin (P < 0.05). After 22 h of IVM BCB+ oocytes showed a prominent chromatin configuration of metaphase I and after 44 h the majority developed a M II nuclear configuration in contrast to the BCB– group (P < 0.0001). Differences were also observed between the two oocyte populations in their mitochondrial activity (P < 0.05). At the beginning of IVM BCB+ oocytes were characterised by high mitochondrial activity in their cytoplasm. The BCB+ oocytes showed clear visible homogenous distributions of mitochondria (P < 0.005) and contained more aggregated clusters of mitochondria in contrast to BCB– oocytes (P < 0.005). In Experiment 2, 318 oocytes were tested for their G6PDH activity and introduced to IVM and IVF. Only oocytes from the BCB+ group, which were matured after 44 h up to the stage of M II (81.6%) were fertilised (17.4%), penetrated (46%) or activated (15.6%) after IVF. These results indicate a relationship between the G6PDH activity of porcine oocytes before IVM and their subsequent nuclear development, mitochondrial activity and aggregation.

199 PREMATURATION OF BOVINE OOCYTES WITH BUTYROLACTONE I AND/OR CILOSTAMIDE: EFFECTS ON MEIOSIS PROGRESSION, CYTOPLASMIC MATURATION AND GENE EXPRESSION

2012 ◽  
Vol 24 (1) ◽  
pp. 212
Author(s):  
G. Z. Mingoti ◽  
F. Filion ◽  
P. Vincent ◽  
L. C. Smith
Keyword(s):  
Cell Communication ◽  
Phosphodiesterase Inhibitor ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Additional Time ◽  
Chi Square ◽  
Developmental Potential ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Meiotic block during a prematuration culture (pre-IVM) before in vitro maturation (IVM) is suggested as a way to provide additional time to synchronize oocyte-somatic cell communication, leading to improved cytoplasmic maturation and nuclear meiotic competence and the acquisition of critical cellular functions necessary for developmental competence. This study was designed to evaluate the effects of the inhibitors butyrolactone I (Bl-I) and cilostamide on oocyte nuclear and cytoplasmic maturation. For that, 2 well-established methods of pre-IVM and IVM for cilostamide (Albuz et al. 2010 Hum. Reprod. 25, 2999–3011) or Bl-I (Hashimoto et al. 2002 Biol Reprod. 66, 1696–1701) were used. Abattoir-collected oocytes were IVM in maturation medium (MM: mSOF with 0.8% BSA and hormones) for 24 h, without a previous pre-IVM culture (control). Cilostamide-group oocytes were treated for the first 2 h in vitro (pre-IVM) with 100 μM of the adenylate cyclase activator forskolin and 500 μM IBMX (nonspecific phosphodiesterase inhibitor) and then oocytes underwent extended IVM for 30 h in the presence of 20 μM cilostamide (type 3 phosphodiesterase inhibitor; pre-IVM 2 h + IVM 30 h). The Bl-I-group oocytes were pre-IVM for 24 h with 100 μM Bl-I diluted in TCM-199 supplemented with 0.2 mM pyruvate and then were IVM in MM for 20 h (pre-IVM 24 h + IVM 20 h). The Bl-I + Cilost group was a combination of both procedures: oocytes were first pre-IVM with Bl-I for 24 h and then cultured as described for the cilostamide group (pre-IVM 24 h + IVM 30 h). Cultures were carried out at 38.5°C in 5% CO2 in humidified air. After IVM, oocytes were stained with 500 nM mitotracker red to assess the mitochondrial membrane potential (Δψm) and with 10 μg mL–1 of Hoescht 33342 to evaluate the nuclear maturation (n = 207). Images were captured by an Olympus confocal microscope and analyzed with Fluoview software. The TUNEL assay was used to detect oocyte DNA fragmentation (n = 74). Relative amounts of mRNA for apoptotic-related genes were quantified after IVM in individual oocytes using real-time PCR (Kameyama et al. 2007 Reproduction 133, 423–32). Means were compared by ANOVA and Tukey's test or by chi-square (P ≤ 0.05). The percentage of oocytes reaching metaphase II after IVM did not differ among groups (73.8 to 90.4%; P ≥ 0.05), indicating that in vitro meiotic resumption was normal. The Δψm, expressed in arbitrary units of fluorescence, was 1.0 ± 0.1a (control), 2.7 ± 0.4b (Bl-I), 3.2 ± 0.5b (Cilost) and 2.1 ± 0.3ab (Bl-I + Cilost). The percentage of TUNEL-positive oocytes (18.8–41.3%) did not differ among groups (P ≥ 0.05). The relative abundance of BAX (1.0 ± 0.4 to 2.3 ± 0.4) and BCL-XL (1.0 ± 0.3 to 0.3 ± 0.1) transcripts was unaffected by pre-IVM and IVM (P ≥ 0.05). In conclusion, except for an increase in mitochondrial activity, pre-IVM with cilostamide and/or Bl-I did not affect cytoplasmic and nuclear oocyte maturation. However, oocyte developmental potential needs to be better evaluated in a future study through assessment of embryonic development. We acknowledge FAPESP and NSERC.

357 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF BOVINE OOCYTES AND THEIR SURROUNDING FOLLICULAR CELLS IN SUBJECT TO THEIR DEVELOPMENTAL COMPETENCE

2010 ◽  
Vol 22 (1) ◽  
pp. 335
Author(s):  
H. Torner ◽  
D. Janowski ◽  
N. Ghanem ◽  
D. Salilew-Wondim ◽  
H. Alm ◽  
...  
Keyword(s):  
Gene Expression ◽  
Caspase 3 ◽  
Expression Profiles ◽  
Developmental Competence ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Altered Expression ◽  
Developmental Potential ◽  
Expression Of Genes ◽  
Bovine Oocytes ◽  
Oocyte Selection

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment. This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).

scholarly journals The embryonic stress response to in vitro culture: insight from genomic analysis

Reproduction ◽  
2016 ◽  
Vol 152 (6) ◽  
pp. R247-R261 ◽  
Author(s):  
Gael Cagnone ◽  
Marc-André Sirard
Keyword(s):  
Stress Responses ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Genomic Analysis ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Mitochondrial Activity ◽  
Long Term Effects ◽  
The Impact

Recent genomic studies have shed light on the impact of in vitro culture (IVC) on embryonic homeostasis and the differential gene expression profiles associated with lower developmental competence. Consistently, the embryonic stress responses to IVC conditions correlate with transcriptomic changes in pathways related to energetic metabolism, extracellular matrix remodelling and inflammatory signalling. These changes appear to result from a developmental adaptation that enhances a Warburg-like effect known to occur naturally during blastulation. First discovered in cancer cells, the Warburg effect (increased glycolysis under aerobic conditions) is thought to result from mitochondrial dysfunction. In the case of IVC embryos, culture conditions may interfere with mitochondrial maturation and oxidative phosphorylation, forcing cells to rely on glycolysis in order to maintain energetic homeostasis. While beneficial in the short term, such adaptations may lead to epigenetic changes with potential long-term effects on implantation, foetal growth and post-natal health. We conclude that lessening the detrimental effects of IVC on mitochondrial activity would lead to significantly improved embryo quality.

160 ZONA PELLUCIDA BIREFRINGENCE CORRELATES WITH CUMULUS MORPHOLOGY AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY OF EQUINE OOCYTES

2012 ◽  
Vol 24 (1) ◽  
pp. 192 ◽  
Author(s):  
A. Mohammadi-Sangcheshmeh ◽  
E. Held ◽  
D. Tesfaye ◽  
K. Schellander ◽  
M. Hoelker
Keyword(s):  
Light Microscopy ◽  
Zona Pellucida ◽  
Polarized Light ◽  
Polarized Light Microscopy ◽  
G6pd Activity ◽  
Developmental Competence ◽  
G6pdh Activity ◽  
Gentamicin Sulfate ◽  
Developmental Capacity ◽  
Glucose 6 Phosphate Dehydrogenase

The present study was conducted to determine the predictive value of zona pellucida birefringence (ZPB) measurement for developmental capacity of equine oocytes. In vitro developmental capacity of equine oocytes is related to cumulus morphology as well as glucose-6-phosphate dehydrogenase (G6PD) activity. Therefore, we classified equine oocytes according to cumulus morphology and G6PDH activity into groups of different developmental capacities and analysed the correlation between ZPB measured by polarized light microscopy and developmental competence. Ovaries were obtained from an abattoir and were transported to the laboratory within 2 to 4 h at 25 to 30°C in 0.9% saline. Cumulus–oocyte complexes (COC) were recovered by scraping the granulosa layer from opened follicles using a bone curette and were classified as having an expanded (Ex, n = 86) or compact (Cp, n = 93) cumulus. Other COC were subjected to 26 μM brilliant cresyl blue (BCB) in modified PBS for 90 min at 38.5°C and were categorised into BCB+ (blue cytoplasm, low G6PD activity, n = 89) and BCB– (colourless cytoplasm, high G6PD activity, n = 41) according to their ability to convert the BCB stain from blue to colourless. Following maturation for 28 to 30 h at 38.5°C in 5% CO2 in DMEM-F12 supplemented with 10% FCS, 5 mU mL–1 of FSH and 25 μg mL–1 of gentamicin sulfate, oocytes were imaged individually to assess zona thickness and ZPB by polarized light microscopy using OCTAX polarAIDE software (OCTAX, Bruckberg, Germany). Subsequently, oocytes were subjected to intracytoplasmic sperm injection using frozen-thawed stallion spermatozoa followed by culture in DMEM/F-12 medium (10% FCS and 25 μg mL–1 of gentamicin sulfate) under mineral oil (38.2°C, 5% CO2, 5% O2) for 8 days. Development rates were compared by chi-square test; variations in thickness and birefringence were analysed by Tukey's test and ANOVA. Higher maturation (58.4 vs 40.2%) and blastocyst rates (11.9 vs 3.8%) of Ex oocytes compared with Cp oocytes as well as higher proportions of BCB+ oocytes that matured (57.7% vs 28.1%), cleaved (45.9 vs 29.0%) and developed to the blastocyst stage (9.2 vs 1.4%) compared with BCB– oocytes (all P < 0.05) confirmed cumulus morphology and G6PDH activity as useful predictors for viability. Moreover, Ex oocytes had a thicker zona (18.2 ± 2.2 μm vs 17.3 ± 2.1 μm) and a higher birefringence (64.6 ± 5.2 vs 62.1 ± 4.2) compared with Cp oocytes (both P < 0.05). Concurrently, oocytes classified as BCB+ had a thicker zona (18.8 ± 2.4 μm vs 16.1 ± 2.0 μm) and higher values for birefringence (63.1 ± 4.5 vs 61.3 ± 3.3) compared with BCB– oocytes (both P < 0.05). Taken together, our results revealed that equine oocytes with higher developmental capacity have a thicker zona and greater birefringence scores compared with oocytes of lower developmental competence. Therefore, ZPB measurement provides a new, noninvasive method to assess the developmental competence of equine oocytes.

scholarly journals Differences in the transcriptional profiles of human cumulus cells isolated from MI and MII oocytes of patients with polycystic ovary syndrome

Reproduction ◽  
2013 ◽  
Vol 145 (6) ◽  
pp. 597-608 ◽  
Author(s):  
Xin Huang ◽  
Cuifang Hao ◽  
Xiaofang Shen ◽  
Xiaoyan Liu ◽  
Yinghua Shan ◽  
...  
Keyword(s):  
Polycystic Ovary Syndrome ◽  
Polycystic Ovary ◽  
Expression Profiles ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Embryo Viability ◽  
Ovary Syndrome ◽  
Developmental Potential ◽  
Qrt Pcr

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. The abnormalities of endocrine and intra-ovarian paracrine interactions may change the microenvironment for oocyte development during the folliculogenesis process and reduce the developmental competence of oocytes in PCOS patients who are suffering from anovulatory infertility and pregnancy loss. In this microenvironment, the cross talk between an oocyte and the surrounding cumulus cells (CCs) is critical for achieving oocyte competence. The aim of our study was to investigate the gene expression profiles of CCs obtained from PCOS patients undergoing IVF cycles in terms of oocyte maturation by using human Genome U133 Plus 2.0 microarrays. A total of 59 genes were differentially expressed in two CC groups. Most of these genes were identified to be involved in one or more of the following pathways: receptor interactions, calcium signaling, metabolism and biosynthesis, focal adhesion, melanogenesis, leukocyte transendothelial migration, Wnt signaling, and type 2 diabetes mellitus. According to the different expression levels in the microarrays and their putative functions, six differentially expressed genes (LHCGR, ANGPTL1, TNIK, GRIN2A, SFRP4, and SOCS3) were selected and analyzed by quantitative RT-PCR (qRT-PCR). The qRT-PCR results were consistent with the microarray data. Moreover, the molecular signatures (LHCGR, TNIK, and SOCS3) were associated with developmental potential from embryo to blastocyst stage and were proposed as biomarkers of embryo viability in PCOS patients. Our results may be clinically important as they offer a new potential strategy for competent oocyte/embryo selection in PCOS patients.

scholarly journals Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early during Neuronal Differentiation

Biology ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 609
Author(s):  
Nunzia Mollo ◽  
Matteo Esposito ◽  
Miriam Aurilia ◽  
Roberta Scognamiglio ◽  
Rossella Accarino ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Mitochondrial Dysfunction ◽  
Neuronal Differentiation ◽  
Expression Profiles ◽  
Meta Analysis ◽  
Atp Synthesis ◽  
Differentiation Markers ◽  
Differentiation Potential ◽  
Atp Production ◽  
Mitochondrial Alterations

Background: The presence of mitochondrial alterations in Down syndrome suggests that it might affect neuronal differentiation. We established a model of trisomic iPSCs, differentiating into neural precursor cells (NPCs) to monitor the occurrence of differentiation defects and mitochondrial dysfunction. Methods: Isogenic trisomic and euploid iPSCs were differentiated into NPCs in monolayer cultures using the dual-SMAD inhibition protocol. Expression of pluripotency and neural differentiation genes was assessed by qRT-PCR and immunofluorescence. Meta-analysis of expression data was performed on iPSCs. Mitochondrial Ca2+, reactive oxygen species (ROS) and ATP production were investigated using fluorescent probes. Oxygen consumption rate (OCR) was determined by Seahorse Analyzer. Results: NPCs at day 7 of induction uniformly expressed the differentiation markers PAX6, SOX2 and NESTIN but not the stemness marker OCT4. At day 21, trisomic NPCs expressed higher levels of typical glial differentiation genes. Expression profiles indicated that mitochondrial genes were dysregulated in trisomic iPSCs. Trisomic NPCs showed altered mitochondrial Ca2+, reduced OCR and ATP synthesis, and elevated ROS production. Conclusions: Human trisomic iPSCs can be rapidly and efficiently differentiated into NPC monolayers. The trisomic NPCs obtained exhibit greater glial-like differentiation potential than their euploid counterparts and manifest mitochondrial dysfunction as early as day 7 of neuronal differentiation.

scholarly journals TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR) Is Upregulated in Lymphocytes Stimulated with Concanavalin A

2021 ◽  
Vol 22 (14) ◽  
pp. 7436
Author(s):  
Helga Simon-Molas ◽  
Xavier Vallvé-Martínez ◽  
Irene Caldera-Quevedo ◽  
Pere Fontova ◽  
Claudia Arnedo-Pac ◽  
...  
Keyword(s):  
Concanavalin A ◽  
Carbon Flux ◽  
Human Lymphocytes ◽  
Tumor Development ◽  
Pentose Phosphate ◽  
Akt Signaling Pathway ◽  
G6pdh Activity ◽  
Physiological Conditions ◽  
Glucose 6 Phosphate Dehydrogenase

The glycolytic modulator TP53-Inducible Glycolysis and Apoptosis Regulator (TIGAR) is overexpressed in several types of cancer and has a role in metabolic rewiring during tumor development. However, little is known about the role of this enzyme in proliferative tissues under physiological conditions. In the current work, we analysed the role of TIGAR in primary human lymphocytes stimulated with the mitotic agent Concanavalin A (ConA). We found that TIGAR expression was induced in stimulated lymphocytes through the PI3K/AKT pathway, since Akti-1/2 and LY294002 inhibitors prevented the upregulation of TIGAR in response to ConA. In addition, suppression of TIGAR expression by siRNA decreased the levels of the proliferative marker PCNA and increased cellular ROS levels. In this model, TIGAR was found to support the activity of glucose 6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentose phosphate pathway (PPP), since the inhibition of TIGAR reduced G6PDH activity and increased autophagy. In conclusion, we demonstrate here that TIGAR is upregulated in stimulated human lymphocytes through the PI3K/AKT signaling pathway, which contributes to the redirection of the carbon flux to the PPP.

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