scholarly journals 114 PORCINE EMBRYO FRAGMENTATION, DEVELOPMENT AND APOPTOSIS: A CONFOCAL MICROSCOPY STUDY

2005 ◽  
Vol 17 (2) ◽  
pp. 207
Author(s):  
B. Mateusen ◽  
A. Van Soom ◽  
D. Maes ◽  
A. de Kruif
Keyword(s):  
Confocal Microscopy ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Morula Stage ◽  
Embryonic Arrest

The relationship between embryonic fragmentation, embryonic arrest, and apoptosis has been the subject of some controversy (Hardy K 1999 Rev. Reprod. 4, 125–134). In order to investigate possible links, in vivo-produced, in vitro-cultured porcine embryos (n = 132) were scored for developmental stage and fragmentation at 7 days post insemination (dpi) and processed for propidium iodide and annexin V labelling. After fixation, embryos were processed for terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Using confocal microscopy, a cell was categorized apoptotic if (i) it had a fragmented or condensed nucleus, (ii) the cell membrane was annexin V-positive, and (iii) the nucleus was TUNEL labelled. An apoptotic cell ratio (ACR) was determined as the percentage of apoptotic cells per embryo. Differences in the % of fragmented and apoptotic embryos and correlations were analyzed using chi-square. Logistic regression was used to compare the average fragmentation % and the ACR. Sixty-one embryos (46%) arrested during the culture period, with 8 embryos arresting before or at the 4-cell stage. Significantly more arrested embryos were fragmented compared to embryos that were blastocysts at 7 dpi. Also, the average fragmentation percentage was significantly higher for arrested embryos compared to blastocysts. The correlation detected between developmental arrest and fragmentation was 0.60 (P < 0.05). None of the embryos without fragmentation had cells categorized as apoptotic, whereas 50 out of 55 embryos with fragmentation possessed apoptotic cells, which led to a correlation of 0.87 (P < 0.01) between fragmentation and apoptosis. The percentage of embryos with apoptotic cells was significantly higher for embryos arrested during the 5-cell to the morula stage compared to embryos that arrested before or at the 4-cell stage and embryos with blastocyst development at 7 dpi. The average ACR of embryos arrested during the 5-cell to the morula stage was significantly higher compared to the average ACR of blastocysts at 7 dpi. The correlation detected between the developmental arrest, during the 5-cell to the morula stage period and apoptosis was 0.57 (P < 0.01). Taken together, significant correlations between fragmentation, developmental arrest and apoptosis were detected. However, the association between embryonic arrest and apoptosis could be established only for embryos arrested after embryonic genome activation.

Quantitative Tumor Apoptosis Imaging Using Technetium-99m–HYNIC Annexin V Single Photon Emission Computed Tomography

2003 ◽  
Vol 21 (18) ◽  
pp. 3483-3487 ◽  
Author(s):  
Christophe Van de Wiele ◽  
Christophe Lahorte ◽  
Hubert Vermeersch ◽  
D. Loose ◽  
Kris Mervillie ◽  
...  
Keyword(s):  
Computed Tomography ◽  
Head And Neck ◽  
Tumor Volume ◽  
Photon Emission ◽  
Annexin V ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Emission Computed Tomography ◽  
Apoptotic Cells ◽  
Tumor Uptake ◽  
Technetium 99M

Purpose: Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m– (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. Patients and Methods: Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. Results: Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P = .012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. Conclusion: In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.

The Role of Urokinase Receptor (uPAR) In Efferocytosis: uPAR Engulfs Apoptotic Cells via a Phosphatidylserine Pathway Mediated by Plasma High Molecular Weight Kininogen

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 929-929 ◽  
Author(s):  
Aizhen Yang ◽  
Jihong Dai ◽  
Raymond B. Birge ◽  
Yi Wu
Keyword(s):  
Molecular Weight ◽  
Flow Cytometry ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Apoptotic Cells ◽  
High Molecular Weight Kininogen ◽  
Viable Cells

Abstract Abstract 929 Phagocytosis of apoptotic cells by phagocytes, also known as efferocytosis, is essential for maintaining normal tissue homeostasis and regulating immune responses. Defects in rapid clearance of apoptotic cells lead to the release of immunogenic cellular contents, which may cause tissue damage and autoimmune disease. Phagocytic receptors differentiate apoptotic cells from viable cells by recognizing ‘don't eat- or eat-me’ signals on the cell surface. Recently, we and others have reported the role of uPAR in mediating efferocytosis. In this study, we examined the mechanism by which uPAR recognizes and internalizes apoptotic cells. By flow cytometry-based in vivo and in vitro phagocytosis assay, we found that in knockout mice the lack of uPAR expression on macrophages decreased their apoptotic cell engulfing activity by >35%. Conversely, soluble uPAR and polyclonal anti-uPAR antibodies (Ab) suppressed the internalization of apoptotic cells by macrophages. However, there was no defect in uPAR-/- macrophage uptake of viable cells, suggesting that uPAR plays a specific role in phagocytosis of apoptotic cells. We established a HEK 293 cell line expressing human full-length uPAR (293-uPAR). In these cells, uPAR-mediated phagocytosis of apoptotic cells was completely blocked by annexin V in the presence of calcium. The effect of annexin V was not observed in the absence of calcium, indicating that uPAR internalizes apoptotic cells through a phosphatidylserine pathway. We also found that uPAR-mediated uptake of apoptotic cells was completely prevented under serum-free conditions. To identify plasma proteins that may opsonize the uPAR function, we used immunodepletion method to test three known uPAR-binding proteins, vitronectin, uPA and high molecular weight kininogen (HK). Depletion of HK from serum by a polyclonal anti-HK Ab significantly reduced the engulfment of apoptotic cells by either macrophages or 293-uPAR cells in a co-culture system. In contrast, depletion of vitronectin or uPA from serum had little effect. uPAR is a GPI-anchored protein. Upon sucrose gradient ultracentrifugation, the majority of uPAR molecules were co-localized with membrane-bound HK in lipid rafts. The binding capacity of HK to apoptotic cell surface was further analyzed by flow cytometry. Phycoerythrin-labeled HK bound to apoptotic cells in a concentration-dependent manner, saturating at 300 nM. In contrast, HK did not bind to viable cells at concentrations up to 1200 nM. It is known that HK is a key component of the plasma contact system and that apoptotic cells potentiate factor Xa formation. Our new findings of the uPAR-HK-phosphatidylserine axis in efferocytosis suggest that this pathway may modulate the coagulation cascade on the surface of apoptotic cells. This pathway may also play a role in the pathogenesis of autoimmune and thrombotic disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development

2009 ◽  
Vol 57 (8) ◽  
pp. 721-730 ◽  
Author(s):  
Andreja Erman ◽  
Daša Zupančič ◽  
Kristijan Jezernik
Keyword(s):  
Apoptotic Cell ◽  
Light And Electron Microscopy ◽  
Tissue Remodeling ◽  
Annexin V ◽  
Cell Loss ◽  
Postnatal Period ◽  
Cell Junctions ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Urothelial Cells ◽  
Junction Protein

Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell–cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI- apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development.

scholarly journals 272OVUM RECOVERY AND BLASTOCYST DEVELOPMENT FOLLOWING INTRACYTOPLASMIC SPERM INJECTION IN CHIMPANZEES

2004 ◽  
Vol 16 (2) ◽  
pp. 256
Author(s):  
I. Hayasaka ◽  
N. Yoshimoto ◽  
Y. Mori ◽  
K. Suzuki ◽  
R. Honda ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Early Embryogenesis ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Morula Stage ◽  
The One ◽  
Estradiol Levels

In the present study, we report on oocyte collection, intracytoplasmic sperm injection and early embryogenesis in chimpanzees. Eight adult female chimpanzees, 11–27 years of age, received a single s.c. injection of 3.75mg GnRH (Leuplin, Takeda Co. Ltd., Osaka, Japan) 1 to 3 days after the beginning of menstruation. Daily i.m. injections of hMG (Humegon, Nippon Organon K.K., Tokyo, Japan) were initiated the following day. The dose of hMG was altered from 75 to 300IU according to serum estradiol levels. When at least one follicle of 17mm or more in diameter was observed, 10000IU of hCG (Pregnyl, Nippon Organon K.K.) were administered by i.m injection. Oocytes were recovered by ultrasound-guided transvaginal follicular aspiration 30.5 to 35.5h after hCG injection. Mature oocytes were denuded of cumulus cells by treatment with 0.1% hyaluronidase, and injected with a frozen-thawed or fresh spermatozoan using a Piezo-driven micromanipulator. Zygotes were cultured in Quinn’s Advantage Fertilization Medium (Cooper Surgical, Inc., Trumbull, CT, USA) with 10 serum protein substitute (SPS) at 37°C in a 5% CO2 atmosphere until the pronucleus stage. The medium was replaced by Quinn’s Advantage Cleavage Medium with 10 SPS from the pronuclear to 8-cell stage, and Quinn’s Advantage Blastcyst Medium with 10 SPS, thereafter. Mild ovarian hyperstimulation syndrome (OHSS) occurred in one female chimpanzee with estradiol levels of 7520pgmL−1. No oocytes were collected from 2 chimpanzees in which large follicles were observed. Thirty-five mature oocytes, one immature oocyte and 6 degenerate/fragmented oocytes were retrieved from 6 chimpanzees, including the one with OHSS. Among 35 mature oocytes injected with spermatozoa, 26 oocytes (74%) produced two pronuclei;; 23 zygotes (66%) cleaved to the 2-cell stage, 22 (63%) to the 4-cell stage, 14 (40%) to the 8-cell stage, and 9 (26%) to the morula stage. Seven zygotes (20%) developed to the blastocyst stage by 120h. There were no differences in fertilization rate or early embryogenesis between frozen and fresh spermatozoa. Results indicate that techniques used for human-assisted reproduction may be applicable to the chimpanzee to help preserve this endangered species.

225 ANTI-APOPTOTIC EFFECT OF AGGREGATION ON PREIMPLANTATION DEVELOPMENT OF BOVINE IN VITRO-FERTILIZED EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 210 ◽  
Author(s):  
S. W. Yoon ◽  
C. H. Park ◽  
S. G. Lee ◽  
H. M. Kim ◽  
J. K. Park ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Mrna Levels ◽  
Cell Stage ◽  
Significant Difference ◽  
Apoptotic Effect

Apoptosis occurs during embryonic development, and is related to early embryonic loss. It is important to produce high-quality blastocysts in vitro for research on the establishment of embryonic stem (ES) cells and transgenic animal production. Therefore, our objectives were to compare the anti-apoptotic effect of bovine aggregate v. nonaggregate IVF embryos and to determine whether aggregation could improve the quality of bovine embryos. The cumulus–oocyte complexes were matured for 20–22 h, and the oocytes were fertilized with cryo-preserved bovine sperm using the swim-up method. After removal of the zona pellucida (ZP), three 4-cell-stage embryos (3X) were aggregated by co-culture in an aggregation hole that was made by an aggregation needle on the culture dish. Embryos were cultured either singularly (1X, ZP removed) or in aggregates of three (3X), and IVF intact embryos served as a control. Five days after aggregation, the developmental rate was observed. The numbers of total cells and apoptotic cells were determined by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay using blastocyst-stage embryos. Moreover, the mRNA expression pattern related to apoptosis and embryo quality was verified by real-time PCR of the aggregated (3X) and nonaggregated (1X) embryos (at least 3 embryos). The percentage of blastocysts was higher in the 3X aggregated embryos (41.3%) compared with that of the 1X ZP-free embryos (24.3%), whereas there was no significant difference in the 1X embryos and the intact controls (24.3 and 25.8%, respectively; P < 0.05). The total cell number of blastocysts also increased approximately threefold (P < 0.05) in 3X aggregated embryos compared with that of 1X controls. In contrast, the percentage of TUNEL-positive cells, an indication of apoptotic cells, was decreased by approximately threefold in 3X aggregated embryos when compared with that of 1X embryos (7.7 and 2.6%, respectively). The mRNA levels for the Oct-4, NANOG, and bcl-2 genes were higher (P < 0.05) and for the Bax gene were lower in the 3X aggregated embryos than for those of the 1X controls. Therefore, our results indicated that aggregation of bovine IVF embryos at a 4-cell stage could promote the quality and suppress the apoptosis of bovine pre-implantation-stage embryos produced in vitro. Further studies are required to investigate the quality of the aggregated embryos in terms of increasing the establishment rate of ES cell lines by seeding on the feeder layer and raising the efficiency of embryo transfer. This work was supported by the BioGreen 21 Program (#20070401034031, #20080401034031), Rural Development Administration, Republic of Korea (HK).

scholarly journals TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement

1998 ◽  
Vol 46 (3) ◽  
pp. 327-334 ◽  
Author(s):  
Françoise Labat-Moleur ◽  
Christiane Guillermet ◽  
Philippe Lorimier ◽  
Catherine Robert ◽  
Sylvie Lantuejoul ◽  
...  
Keyword(s):  
Ph Value ◽  
Apoptotic Cell ◽  
Critical Evaluation ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Detection ◽  
Apoptotic Cells ◽  
Intense Staining ◽  
Tissue Sections ◽  
Enhanced Sensitivity ◽  
Final Answer

TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70–80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

Induction of secondary apoptosis, inflammation, and lung fibrosis after intratracheal instillation of apoptotic cells in rats

2006 ◽  
Vol 290 (4) ◽  
pp. L695-L702 ◽  
Author(s):  
Liying Wang ◽  
James F. Scabilloni ◽  
James M. Antonini ◽  
Yon Rojanasakul ◽  
Vincent Castranova ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Apoptotic Cell ◽  
Death Receptor ◽  
Intratracheal Instillation ◽  
Inflammatory Cells ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Apoptotic Cells ◽  
Lung Pathology ◽  
Continuous Induction

Uncontrolled apoptosis has been associated with several pulmonary disorders; however, the molecular mechanism underlying this process and the fate of apoptotic cells in vivo are unclear. Here we show that direct administration of apoptotic cells to the lungs of rats caused pulmonary inflammation and fibrosis, as indicated by emigration of inflammatory cells to the air spaces, TNF-α immunoreactivity, and connective tissue accumulation, indicating a direct relationship between apoptotic cells and the observed lung pathologies. To determine how the lungs process the accumulated apoptotic cells, normal or apoptotic cells from autologous donor rats were labeled with fluorescent nanobeads and intratracheally instilled into the lungs of rats. Probe distribution and lung cell apoptosis were determined at various times over a 28-day period by confocal fluorescence microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, respectively. Labeled apoptotic cells were cleared by lung macrophages within 1 wk after the treatment. However, the total number of apoptotic cells in the lung remained high at 28 days posttreatment. The results indicate a continuous induction of secondary apoptosis by apoptotic cell instillation, which may contribute to the observed lung pathology. Analysis of lung cell apoptosis by caspase assays showed an elevation of caspase-8 but not caspase-9 in the treatment group at 28 days posttreatment, indicating involvement of the death receptor-mediated pathway in the apoptotic process. Together, our results demonstrate a direct effect of apoptotic cell accumulation on inflammatory and fibrotic pulmonary responses and the continuous induction of lung cell apoptosis by apoptotic cell instillation.

scholarly journals CD74 in Apoptotic Macrophages Is Associated with Inflammation, Plaque Progression and Clinical Manifestations in Human Atherosclerotic Lesions

Metabolites ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 54
Author(s):  
Wei Li ◽  
Nargis Sultana ◽  
Linda Yuan ◽  
Claes Forssell ◽  
Xi-Ming Yuan
Keyword(s):  
Clinical Symptoms ◽  
Plaque Rupture ◽  
Apoptotic Cell ◽  
Clinical Manifestations ◽  
Annexin V ◽  
Statin Treatment ◽  
Terminal Deoxynucleotidyl Transferase ◽  
High Density Lipoproteins ◽  
Atherosclerotic Lesions ◽  
Thrombin Receptors

The aim of this study was to investigate whether CD74 levels in atherosclerotic lesions are associated with inflammation, apoptosis, plaque severity, and clinical symptoms among patients with carotid atherosclerosis. We further studied whether CD74 expression is associated with apoptosis in macrophages induced by 7ketocholesterol (7keto). Sixty-one carotid samples (39 males and 22 females) were immunostained with macrophages, smooth muscle cells, CD74, ferritin, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), and thrombin receptors. Double immunocytochemistry of CD74 and caspase 3 or CD74 and Annexin V was performed on THP-1 macrophages exposed to 7keto. In human carotid plaques, CD74 expression is lesion-dependently increased and is associated with necrotic core formation and plaque rupture, clinical symptoms, macrophage apoptosis, ferritin, and thrombin receptors. CD74 levels were inversely correlated to high-density lipoproteins and statin treatment, and positively correlated to triglycerides. In THP-1 macrophages, 7keto induced a significant increase in levels of CD74, ferritin, and apoptotic cell death. This study suggests that CD74 in apoptotic macrophages is linked to inflammation and thrombosis in progression of human atherosclerotic plaques, lipid metabolism, and clinical manifestation in atherosclerosis. Surface CD74 in apoptotic macrophages and ferritin production induced by oxidized lipids may contribute to inflammation and plaque vulnerability in atherosclerosis.

scholarly journals Maternal Yes-Associated Protein Participates in Porcine Blastocyst Development via Modulation of Trophectoderm Epithelium Barrier Function

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1606 ◽  
Author(s):  
Zubing Cao ◽  
Tengteng Xu ◽  
Xu Tong ◽  
Yiqing Wang ◽  
Dandan Zhang ◽  
...  
Keyword(s):  
Tight Junction ◽  
Lineage Commitment ◽  
Hippo Signaling ◽  
Blastocyst Formation ◽  
Fluid Accumulation ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Downstream Effector ◽  
Morula Stage

The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.

Apoptotic processes and DNA cytosine methylation in mouse embryos arrested at the 2-cell stage

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 269-279 ◽  
Author(s):  
Dušan Fabian ◽  
Alexandra Bukovská ◽  
Štefan Juhás ◽  
Juraj Koppel
Keyword(s):  
Cell Death ◽  
Cytosine Methylation ◽  
Apoptotic Cell ◽  
Annexin V ◽  
Blastocyst Stage ◽  
Nuclear Condensation ◽  
Cell Stage ◽  
Genome Methylation ◽  
Dna Cytosine Methylation

SummaryThe present study evaluates the role of apoptotic cell death and DNA methylation reprogramming in early developmental failures occurring in embryos at the 2-cell stage. Mouse 2-cell embryos were cultured in vitro and treated with chemicals that cause developmental arrest and apoptosis (α-amanitin, actinomycin D, TNF-α). After 24 h, 48 h and 72 h culture, embryos were analysed using cell-death assays (annexin V staining, TUNEL labelling and immunodetection of active caspase-3) and genome methylation assay (immunodetection of 5-methylcytosine). The ability of embryos at the 2-cell stage to undergo apoptotic processes was very low. In arrested embryos, the presence of all evaluated features of apoptosis was recorded only after 72 h culture and their incidence was sporadical. Interestingly, the most frequently observed apoptotic sign was nuclear condensation and the timing of its appearance preceded even the phosphatidylserine flip. Both normally developing and arrested embryos displayed reduction in DNA cytosine methylation. In arrested embryos, this process was independent of cellular cleavage, was more pronounced and finished in almost complete demethylation of the embryonic genome. The timing of the demethylation overlapped with the onset of major apoptotic events. Although observed apoptotic cells showed either demethylated or methylated DNA cytosine in their nuclei, at blastocyst stage the demethylated status appeared more frequently in them.

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