scholarly journals The nucleocytosolic O-fucosyltransferase Spindly affects protein expression and virulence in Toxoplasma gondii

2020 ◽  
pp. jbc.RA120.015883
Author(s):  
Giulia Bandini ◽  
Carolina Agop-Nersesian ◽  
Hanke van der Wel ◽  
Msano Mandalasi ◽  
Hyun W. Kim ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Functional Dependence ◽  
Regulatory Protein ◽  
Protein Accumulation ◽  
Reporter Protein ◽  
Protein Levels ◽  
Endogenous Proteins ◽  
Glcnac Transferase ◽  
Essential Growth

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins, suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.

scholarly journals The nucleocytosolic O-fucosyltransferase Spindly affects protein expression and virulence in Toxoplasma gondii

2020 ◽  
Author(s):  
Giulia Bandini ◽  
Carolina Agop-Nersesian ◽  
Hanke van der Wel ◽  
Msano Mandalasi ◽  
Hyunwoo Kim ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Functional Dependence ◽  
Regulatory Protein ◽  
Protein Accumulation ◽  
Reporter Protein ◽  
Protein Levels ◽  
Endogenous Proteins ◽  
Glcnac Transferase ◽  
Essential Growth

AbstractOnce considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically-expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins, suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.

Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats

2007 ◽  
Vol 292 (5) ◽  
pp. E1456-E1464 ◽  
Author(s):  
Griselda Irusta ◽  
Fernanda Parborell ◽  
Marta Tesone
Keyword(s):  
Direct Action ◽  
Regulatory Protein ◽  
Follicular Development ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Serum Progesterone ◽  
Protein Levels ◽  
Androgen Synthesis ◽  
Cytochrome P 450

Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) α-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly ( P < 0.05), whereas those of androsterone decreased ( P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 α-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant ( P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 α-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.

Increased capacity for sucrose uptake leads to earlier onset of protein accumulation in developing pea seeds

2005 ◽  
Vol 32 (11) ◽  
pp. 997 ◽  
Author(s):  
Elke G. Rosche ◽  
Daniel Blackmore ◽  
Christina E. Offler ◽  
John W. Patrick
Keyword(s):  
Storage Protein ◽  
Protein Biosynthesis ◽  
Protein Bodies ◽  
Protein Accumulation ◽  
Sucrose Uptake ◽  
Wild Type ◽  
Protein Levels ◽  
Vicilin Gene ◽  
And Storage

Pea (Pisum sativum L.) cotyledons, overexpressing a potato sucrose transporter (StSUT1), were used to explore the hypothesis that sucrose stimulates the onset of storage protein biosynthesis. The study focused on the transition between pre-storage and storage phases of seed development. During this period supply of sucrose and hexose to transgenic cotyledons was unaffected by StSUT1 expression. However, protoplasmic levels of sucrose but not hexoses were elevated in transgenic cotyledons. Total protein levels in cotyledons followed the same temporal trend as observed for sucrose and this was reflected in an earlier appearance of protein bodies. Protein levels in wild type and StSUT1 cotyledons were found to lie on the same sucrose dose-response curve and this could be reproduced in vitro when wild type cotyledons were cultured on media containing various sucrose concentrations. Rates of [14C]sucrose uptake and incorporation into polymeric forms were consistent with protoplasmic sucrose supplying a proportion of the carbon skeletons required for storage protein accumulation. In addition, vicilin gene expression was up-regulated earlier in StSUT1 cotyledons. We conclude that sucrose functions both as a signal and fuel to stimulate storage protein accumulation and assembly into protein bodies. An earlier stimulation of storage protein synthesis is considered to largely account for the 14% increase in protein levels of StSUT1 seeds at harvest.

scholarly journals H295R expression of melanocortin 2 receptor accessory protein results in ACTH responsiveness

2015 ◽  
Vol 56 (2) ◽  
pp. 69-76 ◽  
Author(s):  
Kazutaka Nanba ◽  
Andrew X Chen ◽  
Adina F Turcu ◽  
William E Rainey
Keyword(s):  
Cell Line ◽  
Molecular Analysis ◽  
Genetic Manipulation ◽  
Regulatory Protein ◽  
Cellular Protein ◽  
Accessory Protein ◽  
Adrenocortical Cell ◽  
Reading Frame ◽  
Protein Levels

The H295R adrenocortical cell line is widely used for molecular analysis of adrenal functions but is known to have only modest ACTH responsiveness. The lack of ACTH response was linked to a low expression of its receptor, melanocortin 2 receptor (MC2R). We hypothesized that increasing the MC2R accessory protein (MRAP), which is required to traffic MC2R from the endoplasmic reticulum to the cell surface, would increase ACTH responsiveness. Lentiviral particles containing human MRAP-open reading frame were generated and transduced in H295R cells. Using antibiotic resistance, 18 clones were isolated for characterization. The most ACTH-responsive steroidogenic clone, H295RA, was used for further experiments. Successful induction of MRAP and increased expression of MC2R in H295RA cells was confirmed by quantitative real-time RT-PCR and protein analysis. Treatment with ACTH significantly increased aldosterone, cortisol, and dehydroepiandrosterone production in H295RA cells. ACTH also significantly increased transcript levels for all of the steroidogenic enzymes required to produce aldosterone, cortisol, and dehydroepiandrosterone, as well as MC2R mRNA. Using liquid chromatography/tandem mass spectrometry, we further revealed that the main unconjugated steroids produced in H295RA cells were 11-deoxycortisol, cortisol, and androstenedione. Treatment of H295RA cells with ACTH also acutely increased cAMP production and cellular protein levels for total and phosphorylated steroidogenic acute regulatory protein. In summary, through genetic manipulation, we have developed an ACTH-responsive human adrenocortical cell line. The cell line will provide a powerful in vitro tool for molecular analysis of physiologic and pathologic conditions involving the hypothalamic–pituitary–adrenal axis.

scholarly journals IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis

2016 ◽  
Vol 23 (6) ◽  
pp. 481-493 ◽  
Author(s):  
Régia Caroline Peixoto Lira ◽  
Paola Fernanda Fedatto ◽  
David Santos Marco Antonio ◽  
Letícia Ferro Leal ◽  
Carlos Eduardo Martinelli ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Transformation ◽  
Regulatory Protein ◽  
Malignant Cell ◽  
Pathological Features ◽  
Adrenocortical Tumors ◽  
Protein Levels ◽  
Mapk Activity ◽  
High Concentrations

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of theIGF2andIGF1Rgenes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, theIGF2andIGF1Rexpression was evaluated by RT-qPCR according to the patient’s clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. SignificantIGF2overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels ofIGF1Rin patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that theIGF1Roverexpression could be indicative of aggressive ACTs in children. However,in vitrotreatments with high concentrations of OSI-906 (>1μM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.

scholarly journals Identification of Specific Hnrnps As Novel Therapeutic Targets and Responsive Indicators of KPT330 (selinexor) in Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1657-1657 ◽  
Author(s):  
Adam Cloe ◽  
Li Chen ◽  
Yuan Li ◽  
Hongtao Liu ◽  
Jason X. Cheng
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Nuclear Accumulation ◽  
Protein Accumulation ◽  
Clinical Settings ◽  
Bone Marrow Blast ◽  
Protein Levels ◽  
Small Cohort ◽  
Pre Treatment

Abstract Background: Activenuclear-cytoplasmic shuttling of proteins and RNAs, such as heterogeneous ribonucleoproteins (hnRNPs), is essential for the normal function and survival of eukaryotic cells and tumorigenesis (Dreyfuss et al. 1993 Annu Rev Biochem 62, 289; Gorlich and Mattaj 1996 Science 271, 1513). Up-regulation of exportin 1 (XPO1)/chromosomal maintenance 1 (CRM1), a member of the karyopherin-β family of nuclear export receptor proteins, has been implicated in solid and hematologic malignancies (Kau Kau et al. 2004).Selinexor (KPT-330) has been shown to be able block in vitro and in vivo XPO1/CRM1 functions and is currently in phase-II/IIb clinical trials for treatment of hematologic and solid tumors (Senapedis et al., 2014 Nat Rev Cancer 4, 106). However, the mechanisms underlying the selectivity and efficacy of selinexor are incompletely understood, and no biomarkers are currently available to predict clinical responses to selinexor in clinical settings. In this study, we focus on determining the effects of selinexor on the nuclear-cytoplasmic shuttling of hnRNPs, particularly hnRNPK and hnRNPA1, to elucidate the roles of the hnRNPs in the regulation of selectivity and efficacy of selinexor in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Method:We performed growth inhibition/killing assays, histopathologic evaluations, immunohistochemical studies, subcellular fraction western blotting, super-resolution stimulated emission depletion (STED) confocal microcopy and siRNA knockdown experiments. Results: Our in vitro experiments demonstrate a marked increase in XPO1/CRM1 protein and decrease in TP53 in our azacitidine-resistant MDS/AML cell lines compared to our azacitidine-sensitive MDS/AML cell lines. Selinexor treatment efficiently blocks export of hnRNP K from nuclei and increased nuclear accumulation of hnRNPK and inhibits MDS/AML cell growth, while the protein levels of XPO1/CRM1 and TP53 remain unchanged. Our experiments using clinical bone marrow specimens show no significant difference in the total protein level or nuclear accumulation of XPO1/CRM1 between the normal control and MDS or AML bone marrow specimens. In contrast, a strong positive correlation between MDS/AML disease progression and hnRNPK protein accumulation is observed in those clinical specimens. We have extended our experiments to clinical bone marrow specimens from a small cohort in a clinical trial for selinexor in AML at the University of Chicago (NCT02573363). In our small cohort, 5 patients responded to selinexor, 4 patients did not respond and 1 had a partial response. All 5 responders show a striking decrease in their bone marrow blast percentage from their pre-treatment marrows (average blast percentage 37.4%) to their post-treatment (average blast percentage 1.8%). Non-responders show no such difference in pre and post-treatment blast percentage (56.3 and 57.1%, respectively). Importantly, our experiments demonstrate a marked difference in the protein accumulation and subcellular localization of hnRNPK and hnRNPA1, another member of the hnRNP family, between selinexor-responder and selinexor-non-responder bone marrow specimens. Specifically, selinexor responders had much higher levels of hnRNPK and hnRNPA1 proteins in their pre-treatment bone marrows than non-responders, despite the fact that the latter had higher bone marrow blast percentages on average. There is markedly reduced accumulation of hnRNPK and hnRNPA1 in the post-selinexor treatment bone marrow specimens from the responders, but not the non-responders, suggesting these hnRNPs as key therapeutic targets for selinexor in MDS and AML. In contrast, no significant change in XPO1/CRM1 protein levels is observed in the selinexor-responder vs. selinexor-non-responder bone marrow specimens. Conclusion:Our data have revealed a novel drug-action mechanism by which selinexor impairs the nuclear-cytoplasmic shuttling of hnRNPK and hnRNPA1 in MDS and AML cells. Differential expression and localization of these hnRNPs in normal vs. MDS vs. AML cells may provide the rationale for the preferential killing of leukemia cells by selinexor. Our data also suggest the possibility to develop novel hnRNP-based biomarkers to predict the response to selinexor in clinical settings. Disclosures Liu: Karyopharm: Research Funding; BMS: Research Funding.

scholarly journals Translation from the 5′ Untranslated Region (UTR) of mRNA 1 Is Repressed, but That from the 5′ UTR of mRNA 7 Is Stimulated in Coronavirus-Infected Cells

1999 ◽  
Vol 73 (10) ◽  
pp. 8003-8009 ◽  
Author(s):  
Savithra D. Senanayake ◽  
David A. Brian
Keyword(s):  
Viral Gene ◽  
Protein Accumulation ◽  
Regulation Of Transcription ◽  
Reporter Protein ◽  
Virus Growth ◽  
Regulation Of Translation ◽  
Infected Cells ◽  
Made In

ABSTRACT Viral gene products are generally required in widely differing amounts for successful virus growth and assembly. For coronaviruses, regulation of transcription is a major contributor to these differences, but regulation of translation may also be important. Here, we examine the possibility that the 5′ untranslated regions (UTRs), unique for each of the nine species of mRNA in the bovine coronavirus and ranging in length from 70 nucleotides (nt) to 210 nt (inclusive of the common 5′-terminal 65-nt leader), can differentially affect the rate of protein accumulation. When the natural 77-nt 5′ UTR on synthetic transcripts of mRNA 7 (mRNA for N and I proteins) was replaced with the 210-nt 5′ UTR from mRNA 1 (genomic RNA, mRNA for viral polymerase), approximately twofold-less N, or (N) CAT fusion reporter protein, was made in vitro. Twofold less was also made in vivo in uninfected cells when a T7 RNA polymerase-driven transient-transfection system was used. In coronavirus-infected cells, this difference surprisingly became 12-fold as the result of both a stimulated translation from the 77-nt 5′ UTR and a repression of translation from the 210-nt 5′ UTR. These results reveal that a differential 5′ UTR-directed regulation of translation can occur in coronavirus-infected cells and lead us to postulate that the direction and degree of regulation is carried out by viral or virally induced cellular factors acting in trans on cis-acting elements within the 5′ UTR.

scholarly journals Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach

Tumor Biology ◽  
2021 ◽  
Vol 43 (1) ◽  
pp. 309-325
Author(s):  
Yuko Takeba ◽  
Yuki Ohta ◽  
Masanori Ootaki ◽  
Tsukasa Kobayashi ◽  
Keisuke Kida ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Proliferation ◽  
Hepatic Hemangioma ◽  
Protein Accumulation ◽  
Dependent Manner ◽  
Protein Levels ◽  
Erk Phosphorylation ◽  
Tumor Tissues ◽  
Huh7 Cells

BACKGROUND: Cytokines play an important role in the immune response, angiogenesis, cell growth, and differentiation in hepatocellular carcinoma (HCC). OBJECTIVE: We performed a comprehensive study to identify tumor-related cytokines and pathways involved in HCC pathogenesis. METHODS: Cytokine production was evaluated in human HCC tissues and adjacent non-tumor tissues using an antibody-based protein array technique. We compared cytokine expression in HCC tissues with that of hepatic hemangioma (HH), liver metastasis of colorectal cancer, and noncancerous liver tissues from transplantation donors. The protein levels and localization of the candidate cytokines were analyzed by western blotting and immunohistochemistry. RESULTS: Increased expression of interleukin (IL)-1 receptor antagonist, macrophage migration inhibitory factor, and IL-16 was observed in HCC and paired adjacent non-tumor tissues compared with noncancerous livers. In addition, there were increased IL-16 levels in HCC tissues compared with HH. IL-16 treatment significantly increased cell proliferation in vitro. The expression of extracellular signal-regulated kinase (ERK)1/2 and cyclin D1 was markedly increased in cells from two HCC cell lines, Huh7 and HepG2, in a dose- and time-dependent manner. Phosphorylated to total ERK1/2 ratio was increased in Huh7 cells following IL-16 50 ng/ml, but not HepG2 cells. ERK phosphorylation have occurred earlier than protein accumulation at 48 h. Pretreatment with the ERK inhibitor, FR18024, or an anti-IL-16 antibody reduced the increase in IL-16 production in HCC cells. CONCLUSIONS: These results suggest that cell proliferation induced by IL-16 is mediated through the ERK pathway, thus, we identified a new factor associated with HCC tumor growth.

scholarly journals Endothelial SOCS3 maintains homeostasis and promotes survival in endotoxemic mice

2020 ◽  
Author(s):  
Nina Martino ◽  
Ramon Bossardi Ramos ◽  
Shuhan Lu ◽  
Kara Leyden ◽  
Lindsay Tomaszek ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Lethal Dose ◽  
Dose Effect ◽  
Intermediate Phenotype ◽  
Protein Accumulation ◽  
Type I ◽  
Protein Levels ◽  
Function Loss ◽  
Socs3 Protein

AbstractSOCS3 is the main inhibitor of the JAK/STAT3 pathway. This pathway is activated by interleukin 6 (IL-6), a major mediator of the cytokine storm during shock. To determine its role in the vascular response to shock, we challenged mice lacking SOCS3 in the adult endothelium (SOCS3iEKO) with a non-lethal dose of lipopolysaccharide (LPS). SOCS3iEKO mice died 16-24 hours post-injection after severe kidney failure. Loss of SOCS3 led to an LPS-induced type I interferon-like program, and high expression of pro-thrombotic and pro-adhesive genes. Consistently, we observed intraluminal leukocyte adhesion and NETosis, as well as retinal venular leukoembolization. Notably, heterozygous mice displayed an intermediate phenotype, suggesting a gene dose effect. In vitro studies were performed to study the role of SOCS3 protein levels in the regulation of the inflammatory response. In HUVEC, pulse-chase experiments showed that SOCS3 protein has a half-life below 20 minutes. Inhibition of SOCS3 ubiquitination and proteasomal degradation leads to protein accumulation and a stronger inhibition of IL-6 signaling and barrier function loss. Together, our data demonstrates that the regulation of SOCS3 protein levels is critical to inhibit IL-6-mediated endotheliopathy during shock and provides a promising new therapeutic avenue to prevent MODS though stabilization of endothelial SOCS3.Abstract Figure

scholarly journals 3P66SHC, BUT NOT P53, IS INVOLVED IN EARLY ARREST OF IN VITROPRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 124
Author(s):  
L.A. Favetta ◽  
C. Robert ◽  
W.A. King ◽  
D.H. Betts
Keyword(s):  
P53 Protein ◽  
Regulatory Protein ◽  
Bovine Embryo ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Protein Levels ◽  
P53 Mrna

High embryo losses occur in the first week of bovine embryo development, before the activation of the embryonic genome, with a high percentage of embryo death and arrest. Arrested embryos appear morphologically normal and do not exhibit any characteristic sign of apoptosis, including DNA fragmentation. We hypothesized that these embryos enter a senescence-like state and that both the cell cycle regulatory protein p53 and the stress-related protein p66shc, which are involved in the onset of senescence in somatic cells, are responsible for this early embryonic arrest of development. The aim of this study was to characterize the expression of p53 and p66shc in 2–4 cell-arrested bovine embryos. Our experimental model consists of in vitro-produced bovine embryos, co-cultured with oviductal cells. In our in vitro production system 86.8±41.4% of embryos cleave, 13.5±0.5% arrest at the 2–4 cell stage and 24.5±0.7% develop to the blastocyst stage. Cleavage occurs between 26 hours post-insemination (hpi) and 48 hpi. Embryos that cleave by 28hpi show only 0.6±0.3% of 2–4 cell arrest and 41.2±2.1% of blastocyst development, whereas 14.2±0.9% of later-cleaving embryos arrest at the 2–4 cell stage and only 26.5±1.7% develop into blastocysts. We compared 2–4 cell embryos collected at 28hpi with those arrested at the 2–4 cell stage and collected at Day 8 post-insemination. Quantification by Real Time PCR showed significantly higher p66shc mRNA levels (P&lt;0.001), but no changes in p53 mRNA levels (P=0.860) in arrested embryos v. 28-hpi embryos (n=3 pools of 100 embryos each). We obtained the same pattern of p53 and p66shc mRNA expression when we compared 28-hpi embryos with later-cleaving embryos (28hpi to 48hpi, n=3 pools of 100 embryos each), and higher p66shc mRNA levels (P&lt;0.050), or similar p53 mRNA levels (P=0.960). We also confirmed higher p66shc protein levels (P&lt;0.001), but no changes in p53 protein levels (P=1.000), in later cleaving embryos compared with 28hpi-cleaving embryos by semi-quantitative immunocytochemistry (n=70). Statistical analysis was carried out using 2-sample t-test or the equivalent nonparametric test (Mann-Whitney test). Taken together, these results demonstrate that the developmental potential of in vitro-produced embryos is related to the time of first cleavage and that p66shc, but not p53, plays a role in early developmental arrest of in vitro-produced bovine embryos. Further experiments are required to investigate the functional role of p66shc in early embryo arrest. Funded by NSERC, CIHR, OGS and OMAFRA.

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