scholarly journals Single Cell Transcriptomics Reveals Global Markers of Transcriptional Diversity Across Different Forms of Cellular Senescence

2021 ◽  
Author(s):  
Shane A Evans ◽  
Yee Voan Teo ◽  
Kelly Klark ◽  
Takahiro Ito ◽  
John M Sedivy ◽  
...  
Keyword(s):  
Single Cell ◽  
Cellular Senescence ◽  
Replicative Senescence ◽  
Expression Profiles ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Cell Contact ◽  
Matrix Remodeling

Cellular Senescence is a state of irreversible cell cycle arrest, and the accumulation of senescent cells contributes to agerelated organismal decline. The detrimental effects of cellular senescence are due to the senescence associated secretory phenotype (SASP), an array of signaling molecules and growth factors secreted by senescent cells that contribute to the terile inflammation associated with aging tissues. Recent studies, both in vivo and in vitro, have highlighted the heterogeneous nature of the senescence phenotype. In particular, single cell transcriptomics has revealed that Oncogene Induced Senescence (OIS) is characterized by the presence of subpopulations of cells expressing different SASP profiles. We have generated a comprehensive dataset via single-cell transcriptional profiling of genetically homogenous clonal cell lines from different forms of senescence, including OIS, Replicative Senescence (RS), and DNA Damage Induced Senescence (DDIS). We identified subpopulations of cells that are common to all three major forms of senescence and show that the expression profiles of these subpopulations are driven by markers formerly identified in individual forms of senescence. These common signatures are characterized by chromatin modifiers, inflammation, extracellular matrix remodeling, and Ribosomal protein expression. The expression patterns of these subpopulations recapitulate primary and secondary senescence, a phenomenon where a preexisting (primary) senescent cell induces senescence in a neighboring (secondary) cell through cell-to-cell contact. Since it is still unclear what type of senescence occurs in-vivo with age, it is important to know that the formation of primary and secondary populations is common to multiple types of senescence since this mechanism could help explain how senescent cells accumulate in aged organisms. Finally, we show that these subpopulations show differential susceptibility to the senolytic agent Navitoclax, suggesting that senolytic agents targeting the apoptotic pathways may be clearing only a subset of senescent cells based on their inflammatory profiles in-vivo.

scholarly journals Μελέτη της κυτταρικής γήρανσης στον ομαλό λειχήνα του στόματος με τη χρήση της ειδικής χρώσης της λιποφουσκίνης με sudan black B, μιά πρωτότυπη μέθοδο ανίχνευσης γηρασμένων κυττάρων

2014 ◽  
Author(s):  
Ελένη Γεωργακοπούλου
Keyword(s):  
Cellular Senescence ◽  
Replicative Senescence ◽  
Sudan Black B ◽  
Beta Galactosidase

Ως κυτταρική γήρανση (cellular senescence) ορίζεται η μη αναστρέψιμη παύση του κυτταρικού κύκλου συνεπεία είτε εξάντλησης των τελομερών, είτε κυτταρικού στρες, και θεωρείται μηχανισμός αντίστασης στην καρκινογένεση. Το φαινόμενο της κυτταρικής γήρανσης αποτελεί μια ερευνητική πρόκληση μιας και αποτελεί συνδετικό κρίκο μεταξύ της φυσιολογικής γήρανσης της χρόνιας φλεγμονής και βασικών μονοπατιών της καρκινογένεσης. Ο μέχρι σήμερα πιο αξιόπιστος δείκτης κυτταρικής γήρανσης είναι η ανίχνευση της δραστηριότητας της β- γαλακτοσιδάσης των γηρασμένων κυττάρων ,(senescence-associated-beta-galactosidase SA-β-gal). Η μέθοδος αυτή δεν μπορεί να εφαρμοστεί σε αρχειακό υλικό (ιστούς εγκιβωτισμένους σε παραφίνη) αλλά μόνο σε φρέσκους ιστούς και σε τομές από άμεσα κατεψυγμένους ιστούς (κρυοτομές). Εξαιτίας αυτού του περιορισμού υπάρχει έλλειψη εκτενών κλινικοπαθολογικών μελετών για την κυτταρική γήρανση.ΣΚΟΠΟΣ : Επιχειρήθηκε η αναζήτηση ενός βιολογικού δείκτη κυτταρικής γήρανσης, με εφαρμογή σε αρχειακό υλικό. Επίσης, μελετήθηκε η πρωτότυπη χρησιμοποίησή του ως δείκτη κυτταρικής γήρανσης σε ένα χρόνιο φλεγμονώδες νόσημα που αποτελεί μοντέλο συσχέτισης χρόνιας φλεγμονής και καρκίνου, τον Ομαλό λειχήνα του στόματος.ΥΛΙΚΑ ΚΑΙ ΜΕΘΟΔΟΙ : Αναζητώντας μια μέθοδο ανίχνευσης γηρασμένων κυττάρων εφαρμόσιμη σε αρχειακό υλικό, αξιολογήσαμε την ιστοχημική χρώση Sudan-Black B (SBB), που είναι ειδική για την ανίχνευση της λιποφουσκίνης. Η λιποφουσκίνη είναι ένα συσσωμάτωμα οξειδωμένων πρωτεϊνών, λιπιδίων και μετάλλων, η οποία συσσωρεύεται σε γηρασμένους ιστούς. Αναλύσαμε κυτταρικά συστήματα στα οποία προκλήθηκε κυτταρική γήρανση είτε ύστερα από εξάντληση του πολλαπλασιασμού (replicative senescence) ή από στρεσογόνα ερεθίσματα, προ-καρκινικές αλλοιώσεις σε υπό προϋποθέσεις knock-in ποντίκια που παρουσιάζουν γήρανση, και τέλος σε ανθρώπινες προκαρκινικές αλλοιώσεις που γνωρίζουμε ότι περιέχουν γηρασμένα κύτταρα. Η τεχνική εν συνεχεία εφαρμόστηκε σε δείγματα Ομαλού λειχήνα, Ακανθοκυτταρικού καρκινώματος στόματος , καλοήθεις βλάβες στοματικού βλεννογόνου (ινώματα) και φυσιολογικού στοματικού βλεννογόνου.ΑΠΟΤΕΛΕΣΜΑΤΑ : Στα παραπάνω πειράματα αποδείξαμε την συνταύτιση της λιποφουσκίνης και του SA-β-gal σε in vitro και in vivo γηρασμένα κύττταρα (κατεψυγμένους ιστούς). Tα ευρήματα αυτά συνηγορούν πως η λιποφουσκίνη είναι ένας ικανός υποψήφιος δείκτης κυτταρικής γήρανσης. Επιπρόσθετα, κατεψυγμένοι ιστοί θετικοί για SA-β-gal μονιμοποιήθηκαν σε φορμόλη, εγκλείστηκαν σε παραφίνη και βάφτηκαν με SBB. Οι αντίστοιχες SA-β-gal θετικές περιοχές στον ιστό βάφτηκαν ειδικά για λιποφουσκίνη με SBB, ενώ οι ιστοί που ήταν αρνητικοί για SA-β-gal βρέθηκαν και αρνητικοί για τη λιποφουσκίνη. Τα ευρήματα αυτά ενισχύουν περαιτέρω την ευαισθησία και την ειδικότητα της SBB χρώσης για την ανάδειξη γηρασμένων κυττάρων σε αρχειακό υλικό. Η τελευταία μοναδική ιδιότητα του SBB μπορεί να αξιοποιηθεί για κλινικοπαθολογικές μελέτες στο ευρέως διαθέσιμο αρχειακό υλικό. Επιπρόσθετα, η εφαρμογή της τεχνικής σε τομές παραφίνης από Ομαλό λειχήνα, Ακανθοκυτταρικό καρκίνωμα σε ινώματα και φυσιολογικό ιστό στόματος, ανέδειξε την παρουσία γηρασμένων ινοβλαστών στις τομές των ινωμάτων και του Ομαλού λειχήνα και την απουσία τους στο φυσιολογικό ιστό και το Ακανθοκυτταρικό καρκίνωμα του στόματος. Τα ευρήματα αυτά συνηγορούν υπερ μιας προστατευτικής δράσης αυτών των κυττάρων , πιθανά στα πλαίσια μιας καλοήθους αντίδρασης του στρώματος.

Chronic Lymphocytic Leukemia-Associated Macrophages: Not Just Nurse Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 46-46
Author(s):  
Loic Ysebaert ◽  
Mary Poupot ◽  
Yovan Sanchez-Ruiz ◽  
Camille Laurent ◽  
Guy Laurent ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Conditioned Medium ◽  
Apoptotic Cell ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Lymphocytic Leukemia ◽  
Functional Interpretation

Abstract Abstract 46 Introduction: CLL cells interact with many accessory cells in an environment mimicking that of normal mature B cells. Role of antigen, cytokines, adhesion pathways are critical for many aspects in the disease course (proliferation/survival, migration or homing, drug resistance, and presumably relapse). Nurse-like cells (NLC) belong to a monocytic-derived, bystander population among CLL lymph node and spleen stromal cells. Aim: To investigate the nature, functions, and location of NLC within CLL microenvironment. Methods: Gene expression profiles (GEP) from in vitro expanded NLC from patients (n=10) were produced and compared to those from normal CD14+ monocytes, M1-polarized macrophages, M2-polarized macrophages and tumor-associated macrophages (produced in the lab or downloaded from GEO datasets). Principal Component Analysis was used to categorize these five populations of cells and in-house-built GSEA software was used for functional interpretation of their relevant gene lists. Protein expression patterns were validated with multi-analyte ELISArray kits, proteome profiler arrays, flow cytometry (FC) or immunohistochemistry (IHC). Results: New insights into the physiopathological role of NLC in CLL are suggested from five lines of evidence: 1/a Òmonocytic gene signatureÓ (i.e. a set of 549 genes) is shared by the NLC and the monocyte subtypes. The genes over-represented in NLC vs normal monocytes pinpointed positive modulation of apoptotic cell clearance (scavenger, mannose and complement receptors, LXRalpha), lipid metabolism (Apolipoprotein E, PPAR signaling), extracellular matrix-receptor interactions (integrins, SPARC, Matrix MetalloProteinases) and actin cytoskeleton remodeling. 2/unsupervised clustering show that NLC represent an M2-skewed, TAM-like cell population. They down-regulate mRNA and proteins for classic M1 inflammatory markers (e.g. IL-1, IL-6, IL-12, COX2) while increase secretion of TGFbeta, IL-10, CCL17 and CCL22 soluble factors. 3/these and previously published observations suggest that B-CLL-to-NLC interactions may orchestrate immunosuppression in this disease. PBMCs from Òwatch and waitÓ CLL patients (all stage A/Rai 0, mutated IgVH, low risk cytogenetics profile) or healthy donors were stimulated with anti-CD3/CD28 beads + IL-2, either in standard RPMI+10% FCS or in conditioned medium (CM, after 14d CLL-NLC co-culture in vitro) and their proliferation/phenotype were compared after 2 weeks. Significant expansion of T cells with Treg (CD4+CD25+FoxP3+) phenotype was observed only from CLL PBMCs grown in conditioned medium (mean % Treg: 2.85 vs 3.05 in CM for normal PBMCs, and 1.54 vs 15.9 in CM for CLL PBMCs, P< 0.05). 4/although NLC make immune synapses with live B-CLL, they do not phagocytose them. Over-expression of CD47 (ÒdonÕt eat meÓ signal) by B-CLL cells (mfi= 3490 vs 2581 on normal cells, P< 0.05, n=18) may provide them with a protective signal against NLC. 5/from our GEP, flow cytometric and IHC analyses, we propose CD163 (classic M2 marker) as a reliable tool to identify NLC in vivo. Although in vitro, CLL cells can pervert healthy donor monocytes into NLC, only CLL-derived NLC are truly CD14+ CD163+. In vivo, CD163 staining reveals putative NLC in CLL lymph nodes(LN)/spleen sections but not in bone marrow. In LN from all patients, NLC reside in the subcapsular areas and line vessel structures, suggesting a role in CLL cells trafficking. Most interestingly, NLC infiltrate pseudofollicles structures only in a subset of cases. We will present updated IHC and clinical presentation correlation studies. Conclusions: Our results suggest that the role of NLC in CLL might be broader than initially thought. Beside of nursing and conferring drug resistance, NLC may also be crucial in the setting of immunosuppression, of CLL cells recruitment, and should thus be considered as therapeutic targets. Disclosures: Off Label Use: GA101 is not currently approved for CLL treatment.

scholarly journals Phosphorylation and Stabilization of HURP by Aurora-A: Implication of HURP as a Transforming Target of Aurora-A

2005 ◽  
Vol 25 (14) ◽  
pp. 5789-5800 ◽  
Author(s):  
Chang-Tze Ricky Yu ◽  
Jung-Mao Hsu ◽  
Yuan-Chii Gladys Lee ◽  
Ann-Ping Tsou ◽  
Chen-Kung Chou ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Gene Expression Profiles ◽  
Aurora A ◽  
Initial Attempt ◽  
Stable Transfectants ◽  
Low Serum

ABSTRACT Aurora-A, a mitotic serine/threonine kinase with oncogene characteristics, has recently drawn intense attention because of its association with the development of human cancers and its relationship with mitotic progression. Using the gene expression profiles of Aurora-A as a template to search for and compare transcriptome expression profiles in publicly accessible microarray data sets, we identified HURP (encodes hepatoma upregulated protein) as one of the best Aurora-A-correlated genes. Empirical validation indicates that HURP has several characteristics in common with Aurora-A. These two genes have similar expression patterns in hepatocellular carcinoma, liver regeneration after partial hepatectomy, and cell cycle progression and across a variety of tissues and cell lines. Moreover, Aurora-A phosphorylated HURP in vitro and in vivo. Ectopic expression of either the catalytically inactive form of Aurora-A or the HURP-4P mutant, in which the Aurora-A phosphorylation sites were replaced with Ala, resulted in HURP instability and complex disassembly. In addition, HURP-wild-type stable transfectants were capable of growing in low-serum environments whereas HURP-4P grew poorly under low-serum conditions and failed to proliferate. These studies together support the view that the ability to integrate evidence derived from microarray studies into biochemical analyses may ultimately augment our predictive power when analyzing the potential role of poorly characterized proteins. While this combined approach was simply an initial attempt to answer a range of complex biological questions, our findings do suggest that HURP is a potential oncogenic target of Aurora-A.

scholarly journals Developmental and molecular correlates of bovine preimplantation embryos

Reproduction ◽  
2006 ◽  
Vol 131 (5) ◽  
pp. 895-904 ◽  
Author(s):  
Hakan Sagirkaya ◽  
Muge Misirlioglu ◽  
Abdullah Kaya ◽  
Neal L First ◽  
John J Parrish ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methyltransferase ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Preimplantation Embryos ◽  
Developmental Potential

Expression of embryonic genes is altered in different culture conditions, which influence developmental potential both during preimplantation and fetal development. The objective of this study was to define the effects of culture conditions on: bovine embryonic development to blastocyst stage, blastocyst cell number, apoptosis and expression patterns of a panel of developmentally important genes. Bovine embryos were culturedin vitroin three culture media containing amino acids, namely potassium simplex optimization medium (KSOMaa), Charles Rosenkrans 1 (CR1aa) and synthetic oviductal fluid (SOFaa). Apoptosis in blastocysts was determined by TUNEL assay and expression profiles of developmentally important genes were assayed by real-time PCR.In vivo-produced bovine blastocysts were used as controls for experiments determining gene expression patterns. While the cleavage rates did not differ, embryos cultured in SOFaa had higher rates of development to blastocyst stage (P< 0.05). Mean cell numbers and percentages of apoptotic cells per blastocyst did not differ among the groups. Expression of the heat shock protein 70 (Hsp70) gene was significantly up-regulated in both CR1aa and KSOMaa when compared with SOFaa (P< 0.001). DNA methyltransferase 3a (Dnmt3a) expression was higher in embryos cultured in CR1aa than in those cultured in SOFaa (P< 0.001). Expression of interferon tau (IF-τ) and insulin-like growth factor II receptor (Igf-2r) genes was significantly up-regulated in KSOMaa when compared with CR1aa (P< 0.001). Gene expression did not differ betweenin vivo-derived blastocysts and theirin vitro-derived counterparts. In conclusion, SOFaa supports higher development to blastocyst stage than KSOMaa and CR1aa, and the culture conditions influence gene expression.

scholarly journals A systematic dissection of human primary osteoblasts in vivo at single-cell resolution

2020 ◽  
Author(s):  
Yun Gong ◽  
Junxiao Yang ◽  
Xiaohua Li ◽  
Cui Zhou ◽  
Yu Chen ◽  
...  
Keyword(s):  
Bone Formation ◽  
Single Cell ◽  
Bone Cells ◽  
Expression Patterns ◽  
Cellular Heterogeneity ◽  
Human Osteoblasts ◽  
Primary Osteoblasts ◽  
Human Primary Osteoblasts

AbstractOsteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. Although both in vivo and in vitro studies on mice have identified several potential osteoblast subtypes based on their different transition stages or biological responses to external stimuli, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was mainly characterized by the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2), and its existence was confirmed by immunofluorescence staining. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human osteoblasts in vivo and mouse osteoblasts both in vivo and in vitro, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level, which is an important and necessary step to further dissect the biological roles of osteoblasts in bone metabolism under various (patho-) physiological conditions.

scholarly journals Transcriptional Profiling of Bacillus anthracis during Infection of Host Macrophages

2007 ◽  
Vol 75 (7) ◽  
pp. 3434-3444 ◽  
Author(s):  
Nicholas H. Bergman ◽  
Erica C. Anderson ◽  
Ellen E. Swenson ◽  
Brian K. Janes ◽  
Nathan Fisher ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Host Cell ◽  
Transcriptional Profiling ◽  
Expression Patterns ◽  
Survival Strategies ◽  
Reverse Transcription Pcr ◽  
Bacterial Rna ◽  
Definition Of

ABSTRACT The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

scholarly journals Comprehensive Integration of Single-Cell Transcriptional Profiling Reveals the Heterogeneities of Non-cardiomyocytes in Healthy and Ischemic Hearts

2020 ◽  
Vol 7 ◽  
Author(s):  
Lingfang Zhuang ◽  
Lin Lu ◽  
Ruiyan Zhang ◽  
Kang Chen ◽  
Xiaoxiang Yan
Keyword(s):  
Myocardial Infarction ◽  
Single Cell ◽  
Rna Sequencing ◽  
Molecular Mechanisms ◽  
Developmental Trajectories ◽  
Transcriptional Profiling ◽  
Sequencing Data ◽  
Signature Genes

Advances in single-cell RNA sequencing (scRNA-seq) technology have recently shed light on the molecular mechanisms of the spatial and temporal changes of thousands of cells simultaneously under homeostatic and ischemic conditions. The aim of this study is to investigate whether it is possible to integrate multiple similar scRNA-seq datasets for a more comprehensive understanding of diseases. In this study, we integrated three representative scRNA-seq datasets of 27,349 non-cardiomyocytes isolated at 3 and 7 days after myocardial infarction or sham surgery. In total, seven lineages, including macrophages, fibroblasts, endothelia, and lymphocytes, were identified in this analysis with distinct dynamic and functional properties in healthy and nonhealthy hearts. Myofibroblasts and endothelia were recognized as the central hubs of cellular communication via ligand-receptor interactions. Additionally, we showed that macrophages from different origins exhibited divergent transcriptional signatures, pathways, developmental trajectories, and transcriptional regulons. It was found that myofibroblasts predominantly expand at 7 days after myocardial infarction with pro-reparative characteristics. We identified signature genes of myofibroblasts, such as Postn, Cthrc1, and Ddah1, among which Ddah1 was exclusively expressed on activated fibroblasts and exhibited concordant upregulation in bulk RNA sequencing data and in vivo and in vitro experiments. Collectively, this compendium of scRNA-seq data provides a valuable entry point for understanding the transcriptional and dynamic changes of non-cardiomyocytes in healthy and nonhealthy hearts by integrating multiple datasets.

scholarly journals Divergent expression patterns of pituitary gonadotropin subunit and GnRH receptor genes to continuous GnRH in vitro and in vivo

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marija M. Janjic ◽  
Rafael M. Prévide ◽  
Patrick A. Fletcher ◽  
Arthur Sherman ◽  
Kosara Smiljanic ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Reproductive Function ◽  
Gnrh Receptor ◽  
Continuous Treatment ◽  
Pituitary Cells ◽  
Cell Content ◽  
Major Factors

AbstractContinuous, as opposed to pulsatile, delivery of hypothalamic gonadotropin-releasing hormone (GnRH) leads to a marked decrease in secretion of pituitary gonadotropins LH and FSH and impairment of reproductive function. Here we studied the expression profile of gonadotropin subunit and GnRH receptor genes in rat pituitary in vitro and in vivo to clarify their expression profiles in the absence and continuous presence of GnRH. Culturing of pituitary cells in GnRH-free conditions downregulated Fshb, Cga, and Gnrhr expression, whereas continuous treatment with GnRH agonists upregulated Cga expression progressively and Gnrhr and Fshb expression transiently, accompanied by a prolonged blockade of Fshb but not Gnrhr expression. In contrast, Lhb expression was relatively insensitive to loss of endogenous GnRH and continuous treatment with GnRH, probably reflecting the status of Egr1 and Nr5a1 expression. Similar patterns of responses were observed in vivo after administration of a GnRH agonist. However, continuous treatment with GnRH stimulated LH secretion in vitro and in vivo, leading to decrease in LH cell content despite high basal Lhb expression. These data suggest that blockade of Fshb expression and depletion of the LH secretory pool are two major factors accounting for weakening of the gonadotroph secretory function during continuous GnRH treatment.

scholarly journals Effects of different oocyte retrieval and in vitro maturation systems on bovine embryo development and quality

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 367-377 ◽  
Author(s):  
Sandra Milena Bernal ◽  
Julia Heinzmann ◽  
Doris Herrmann ◽  
Bernd Timmermann ◽  
Ulrich Baulain ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Global Methylation ◽  
Oocyte Developmental Competence

SummaryCyclic adenosine monophosphate (cAMP) modulators have been used to avoid spontaneous oocyte maturation and concomitantly improve oocyte developmental competence. The current work evaluated the effects of the addition of cAMP modulators forskolin, 3-isobutyl-1-methylxanthine (IBMX) and cilostamide during in vitro maturation on the quality and yields of blastocysts. The following experimental groups were evaluated: (i) slicing or (ii) aspiration and maturation in tissue culture medium (TCM)199 for 24 h (TCM24slicing and TCM24aspiration, respectively), (iii) aspiration and maturation in the presence of cAMP modulators for 30 h (cAMP30aspiration) and in vivo-produced blastocysts. In vitro-matured oocytes were fertilized and presumptive zygotes were cultured in vitro to assess embryo development. Cleavage, blastocyst formation, blastocyst cell number, mRNA abundance of selected genes and global methylation profiles were evaluated. Blastocyst rate/zygotes for the TCM24aspiration protocol was improved (32.2 ± 2.1%) compared with TCM24slicing and cAMP30aspiration (23.4 ± 1.2% and 23.3 ± 2.0%, respectively, P<0.05). No statistical differences were found for blastocyst cell numbers. The mRNA expression for the EGR1 gene was down-regulated eight-fold in blastocysts that had been produced in vitro compared with their in vivo counterparts. Gene expression profiles for IGF2R, SLC2A8, COX2, DNMT3B and PCK2 did not differ among experimental groups. Bovine testis satellite I and Bos taurus alpha satellite methylation profiles from cAMP30aspiration protocol-derived blastocysts were similar to patterns that were observed in their in vivo equivalents (P > 0.05), while those from the other groups were significantly elevated. It is concluded that retrieval, collection systems and addition of cAMP modulators can affect oocyte developmental competence, which is reflected not only in blastocyst rates but also in global DNA methylation and gene expression patterns.

scholarly journals miR-31 Functions as a Negative Regulator of Lymphatic Vascular Lineage-Specific Differentiation In Vitro and Vascular Development In Vivo

2010 ◽  
Vol 30 (14) ◽  
pp. 3620-3634 ◽  
Author(s):  
Deena M. Leslie Pedrioli ◽  
Terhi Karpanen ◽  
Vasilios Dabouras ◽  
Giorgia Jurisic ◽  
Glenn van de Hoek ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular System ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Vascular Development ◽  
Negative Regulator ◽  
Tissue Fluid ◽  
Specific Differentiation

ABSTRACT The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses, and promotes cancer metastasis. To address the role microRNAs (miRNAs) play in the development and function of the lymphatic vascular system, we defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs) and identified four BVEC signature and two LEC signature miRNAs. Their vascular lineage-specific expression patterns were confirmed in vivo by quantitative real-time PCR and in situ hybridization. Functional characterization of the BVEC signature miRNA miR-31 identified a novel BVEC-specific posttranscriptional regulatory mechanism that inhibits the expression of lymphatic lineage-specific transcripts in vitro. We demonstrate that suppression of lymphatic differentiation is partially mediated via direct repression of PROX1, a transcription factor that functions as a master regulator of lymphatic lineage-specific differentiation. Finally, in vivo studies of Xenopus and zebrafish demonstrated that gain of miR-31 function impaired venous sprouting and lymphatic vascular development, thus highlighting the importance of miR-31 as a negative regulator of lymphatic development. Collectively, our findings identify miR-31 is a potent regulator of vascular lineage-specific differentiation and development in vertebrates.

Sign in / Sign up

Export Citation Format

Share Document