71 WHAT IS THE UTERINE RESPONSE IN A CLONED BOVINE PREGNANCY?

2006 ◽  
Vol 18 (2) ◽  
pp. 144 ◽  
Author(s):  
T. C. Santos ◽  
F. T. V. Pereira ◽  
A. C. Assis Neto ◽  
C. E. Ambrósio ◽  
F. V. Meirelles ◽  
...  
Keyword(s):  
Cell Cycle ◽  
First Trimester ◽  
Giant Cells ◽  
Differential Analysis ◽  
Placental Development ◽  
Trophoblastic Cells ◽  
M Phase ◽  
Cell Cycle Phases ◽  
Cloned Bovine ◽  
Trophoblast Giant Cells

Bovine has a synepitheliochorial placenta and characteristically there is no invasion of the trophoblast, but there is migration of the binucleate trophoblast giant cells into the maternal endometrium. The feto-maternal interface occurs in placentome where a tridimensional organization permits interactions between maternal epithelium and trophoblast, and in the intercaruncular area it is possible to observe a few mini-placentomes and the uterine glands opening. The objective of the present investigation was to study the morphological aspects of the uterus in bovine that had a cloned cattle gestations to understand the differences with natural gestation. The uterus and fetal membranes from natural and cloned cattle gestations were collected, fixed in 10% formaldehyde, processed, and stained for light microscopy and immunohistochemistry. The morphological differences observed in the surrogate uterus were: extensive areas without placentome, hemorrhagic uterine areas, caruncular fusion giving a reduced number of caruncules, increase in size and weight (megacaruncules), and a significant number of mini-caruncules giving miniplacentomes (diameter < 1 cm). In particular the mini-placentome showed functional trophoblastic cells with PAS+ granules in the binucleate trophoblast giant cells and an intense subepithelial capillary organization in maternal and fetal sides. The normal and clone placentomal cell populations were analyzed throughout pregnancy. The population of tetraploid and diploid trophoblastic cells was stained; detached cell cycle and DNA content was measured in FL2 using a FACscalibur flow cytometric system. We determined the percentage of cells in apoptosis (sub-G1), quiescent cells (G0/G1), synthesis (S), and proliferative cells (G2/M) with the aid of ModFit software. In addition, a cell cycle differential analysis was performed, and the tetraploid population presented statistical differences in cell cycle phases and populations relative to the apoptosis rate for the first (7.5 � 3.1%), second (15.2 � 5.0%) and third (17.3 � 4.3%) trimesters. The number of apoptotic cells increased significantly during pregnancy stages. The results showed that first trimester presented the majority of its cells in the G0-G1 phase, starting the cell cycle. On the other hand, the second and third trimesters presented the majority of their cells in the G2-M phase, ending the cell cycle. The relationship between cell cycle phases/rate of apoptosis in mononucleate cells, days of normal and cloned pregnancy, the number of binucleate cells, and their metabolic activity as well as their developmental kinetics could be important data in several studies that involve placental development in natural pregnancy or that derived from laboratory-manipulated embryos. This work was supported by FAPESP and CNPq.

scholarly journals An automaton model for the cell cycle

2010 ◽  
Vol 1 (1) ◽  
pp. 36-47 ◽  
Author(s):  
Atilla Altinok ◽  
Didier Gonze ◽  
Francis Lévi ◽  
Albert Goldbeter
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Deterministic Model ◽  
Initial Conditions ◽  
Cell Number ◽  
Time Step ◽  
M Phase ◽  
Random Manner ◽  
The Mean ◽  
Cell Cycle Phases

We consider an automaton model that progresses spontaneously through the four successive phases of the cell cycle: G1, S (DNA replication), G2 and M (mitosis). Each phase is characterized by a mean duration τ and a variability V . As soon as the prescribed duration of a given phase has passed, the transition to the next phase of the cell cycle occurs. The time at which the transition takes place varies in a random manner according to a distribution of durations of the cell cycle phases. Upon completion of the M phase, the cell divides into two cells, which immediately enter a new cycle in G1. The duration of each phase is reinitialized for the two newborn cells. At each time step in any phase of the cycle, the cell has a certain probability to be marked for exiting the cycle and dying at the nearest G1/S or G2/M transition. To allow for homeostasis, which corresponds to maintenance of the total cell number, we assume that cell death counterbalances cell replication at mitosis. In studying the dynamics of this automaton model, we examine the effect of factors such as the mean durations of the cell cycle phases and their variability, the type of distribution of the durations, the number of cells, the regulation of the cell population size and the independence of steady-state proportions of cells in each phase with respect to initial conditions. We apply the stochastic automaton model for the cell cycle to the progressive desynchronization of cell populations and to their entrainment by the circadian clock. A simple deterministic model leads to the same steady-state proportions of cells in the four phases of the cell cycle.

Phosphatidylcholine catabolism in the MCF-7 cell cycle

2006 ◽  
Vol 84 (5) ◽  
pp. 737-744 ◽  
Author(s):  
Weiyang Lin ◽  
Gilbert Arthur
Keyword(s):  
Cell Cycle ◽  
S Phase ◽  
Specific Cell ◽  
Synchronized Cells ◽  
M Phase ◽  
G2 Phase ◽  
Cell Cycle Phases ◽  
Kinetics Of ◽  
Mcf 7

The catabolism of phosphatidylcholine (PtdCho) appears to play a key role in regulating the net accumulation of the lipid in the cell cycle. Current protocols for measuring the degradation of PtdCho at specific cell-cycle phases require prolonged periods of incubation with radiolabelled choline. To measure the degradation of PtdCho at the S and G2 phases in the MCF-7 cell cycle, protocols were developed with radiolabelled lysophosphatidylcholine (lysoPtdCho), which reduces the labelling period and minimizes the recycling of labelled components. Although most of the incubated lysoPtdCho was hydrolyzed to glycerophosphocholine (GroPCho) in the medium, the kinetics of the incorporation of label into PtdCho suggests that the labelled GroPCho did not contribute significantly to cellular PtdCho formation. A protocol which involved exposing the cells twice to hydroxyurea, was also developed to produce highly synchronized MCF-7 cells with a profile of G1:S:G2/M of 90:5:5. An analysis of PtdCho catabolism in the synchronized cells following labelling with lysoPtdCho revealed that there was increased degradation of PtdCho in early to mid-S phase, which was attenuated in the G2/M phase. The results suggest that the net accumulation of PtdCho in MCF-7 cells may occur in the G2 phase of the cell cycle.

scholarly journals Centrosome Clustering in the Development of Bovine Binucleate Trophoblast Giant Cells

2016 ◽  
Vol 203 (5) ◽  
pp. 287-294 ◽  
Author(s):  
Karl Klisch ◽  
Elisabeth M. Schraner ◽  
Alois Boos
Keyword(s):  
Cell Cycle ◽  
Tumor Cells ◽  
Molecular Mechanisms ◽  
Giant Cells ◽  
Cell Nuclei ◽  
Pericentriolar Material ◽  
A Cell ◽  
Pass Through ◽  
Diploid Cells ◽  
Trophoblast Giant Cells

Binucleate trophoblast giant cells (BNC) are the characteristic feature of the ruminant placenta. During their development, BNC pass through 2 acytokinetic mitoses and become binucleate with 2 tetraploid nuclei. In this study, we investigate the number and location of centrosomes in bovine BNC. Centrosomes typically consist of 2 centrioles surrounded by electron-dense pericentriolar material. Duplication of centrosomes is tightly linked to the cell cycle, which ensures that the number of centrosomes remains constant in proliferating diploid cells. Alterations of the cell cycle, which affect the number of chromosome sets, also affect the number of centrosomes. In this study, we use placentomal tissue from pregnant cows (gestational days 80-230) for immunohistochemical staining of γ-tubulin (n = 3) and transmission electron microscopy (n = 3). We show that mature BNC have 4 centrosomes with 8 centrioles, clustered in the angle between the 2 cell nuclei. During the second acytokinetic mitosis, the centrosomes must be clustered to form the poles of a bipolar spindle. In rare cases, centrosome clustering fails and tripolar mitosis leads to the formation of trinucleate “BNC”. Generally, centrosome clustering occurs in polyploid tumor cells, which have an increased number of centrioles, but it is absent in proliferating diploid cells. Thus, inhibition of centrosome clustering in tumor cells is a novel promising strategy for cancer treatment. BNC are a cell population in which centrosome clustering occurs as part of the normal life history. Thus, they might be a good model for the study of the molecular mechanisms of centrosome clustering.

scholarly journals Maternal Obesity Alters Placental Cell Cycle Regulators in the First Trimester of Human Pregnancy: New Insights for BRCA1

2020 ◽  
Vol 21 (2) ◽  
pp. 468 ◽  
Author(s):  
Denise Hoch ◽  
Martina Bachbauer ◽  
Caroline Pöchlauer ◽  
Francisco Algaba-Chueca ◽  
Veronika Tandl ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Maternal Obesity ◽  
First Trimester ◽  
Cell Cycle Regulators ◽  
Placental Development ◽  
Cell Cycle Protein ◽  
Short Term Treatment ◽  
Placental Cell ◽  
Human Pregnancy ◽  
Wide Range

In the first trimester of pregnancy, placental development involves a wide range of cellular processes. These include trophoblast proliferation, fusion, and differentiation, which are dependent on tight cell cycle control. The intrauterine environment affects placental development, which also includes the trophoblast cell cycle. In this work, we focus on maternal obesity to assess whether an altered intrauterine milieu modulates expression and protein levels of placental cell cycle regulators in early human pregnancy. For this purpose, we use first trimester placental tissue from lean and obese women (gestational week 5+0–11+6, n = 58). Using a PCR panel, a cell cycle protein array, and STRING database analysis, we identify a network of cell cycle regulators increased by maternal obesity in which breast cancer 1 (BRCA1) is a central player. Immunostaining localizes BRCA1 predominantly to the villous and the extravillous cytotrophoblast. Obesity-driven BRCA1 upregulation is not able to be explained by DNA methylation (EPIC array) or by short-term treatment of chorionic villous explants at 2.5% oxygen with tumor necrosis factor α (TNF-α) (50 mg/mL), leptin (100 mg/mL), interleukin 6 (IL-6) (100 mg/mL), or high glucose (25 nM). Oxygen tension rises during the first trimester, but this change in vitro has no effect on BRCA1 (2.5% and 6.5% O2). We conclude that maternal obesity affects placental cell cycle regulation and speculate this may alter placental development.

scholarly journals Reprogramming the Cell Cycle for Endoreduplication in Rodent Trophoblast Cells

1998 ◽  
Vol 9 (4) ◽  
pp. 795-807 ◽  
Author(s):  
Alasdair MacAuley ◽  
James C. Cross ◽  
Zena Werb
Keyword(s):  
Cell Cycle ◽  
Cell Differentiation ◽  
Giant Cell ◽  
Mitotic Cycle ◽  
Mitotic Cell ◽  
Giant Cells ◽  
Cyclin B ◽  
Helix Loop Helix ◽  
Trophoblast Giant Cells ◽  
Cycle Regulation

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34cdk1complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.

scholarly journals Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Xiwen Hu ◽  
Jiangchao Li ◽  
Qianqian Zhang ◽  
Lingyun Zheng ◽  
Guang Wang ◽  
...  
Keyword(s):  
Fetal Death ◽  
Underlying Mechanism ◽  
Giant Cells ◽  
Placental Development ◽  
Genetic Inactivation ◽  
Chorioallantoic Placenta ◽  
Phosphoinositide 3 Kinase ◽  
Trophoblast Giant Cells ◽  
Mammalian Embryonic Development

Abstract Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δD910A/D910A), we show that fetuses carried by p110δD910A/D910A females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δD910A/D910A females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.

scholarly journals Knockdown of Splicing Complex Protein PCBP2 Reduces Extravillous Trophoblast Differentiation Through Transcript Switching

2021 ◽  
Vol 9 ◽  
Author(s):  
Danai Georgiadou ◽  
Souad Boussata ◽  
Remco Keijser ◽  
Dianta A. M. Janssen ◽  
Gijs B. Afink ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hellp Syndrome ◽  
Rna Splicing ◽  
Ex Vivo ◽  
First Trimester ◽  
Cellular Growth ◽  
Extravillous Trophoblast ◽  
Placental Development ◽  
Alternative Mrna Splicing

Mutations in the LINC-HELLP non-coding RNA (HELLPAR) have been associated with familial forms of the pregnancy-specific HELLP syndrome. These mutations negatively affect extravillous trophoblast (EVT) differentiation from a proliferative to an invasive state and disturb the binding of RNA splicing complex proteins PCBP1, PCBP2, and YBX1 to LINC-HELLP. In this study, by using both in vitro and ex vivo experiments, we investigate if these proteins are involved in the regulation of EVT invasion during placentation. Additionally, we study if this regulation is due to alternative mRNA splicing. HTR-8/SVneo extravillous trophoblasts and human first trimester placental explants were used to investigate the effect of siRNA-mediated downregulation of PCBP1, PCBP2, and YBX1 genes on the differentiation of EVTs. Transwell invasion assays and proliferation assays indicated that upon knockdown of PCBP2 and, to a lesser extent, YBX1 and PCBP1, EVTs fail to differentiate toward an invasive phenotype. The same pattern was observed in placental explants where PCBP2 knockdown led to approximately 80% reduction in the number of explants showing any EVT outgrowth. Of the ones that still did show EVT outgrowth, the percentage of proliferating EVTs was significantly higher compared to explants transfected with non-targeting control siRNAs. To further investigate this effect of PCBP2 silencing on EVTs, we performed whole transcriptome sequencing (RNA-seq) on HTR-8/SVneo cells after PCBP2 knockdown. PCBP2 knockdown was found to have minimal effect on mRNA expression levels. In contrast, PCBP2 silencing led to a switch in splicing for a large number of genes with predominant functions in cellular assembly and organization, cellular function and maintenance, and cellular growth and proliferation and the cell cycle. EVTs, upon differentiation, alter their function to be able to invade the decidua of the mother by changing their cellular assembly and their proliferative activity by exiting the cell cycle. PCBP2 appears to be a paramount regulator of these differentiation mechanisms, where its disturbed binding to LINC-HELLP could contribute to dysfunctional placental development as seen in the HELLP syndrome.

Flow Cytometric Quantification of All Cell Cycle Phases and Apoptosis in a Two-Color Fluorescence Plot

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4720-4720
Author(s):  
Olivier Herault ◽  
Christine Vignon
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Histone H3 ◽  
Leukemic Cells ◽  
Becton Dickinson ◽  
Alexa Fluor ◽  
Flow Cytometric ◽  
M Phase ◽  
Cell Cycle Phases ◽  
The Impact

Abstract Abstract 4720 An optimal technology for cell cycle analysis would allow measuring concomitantly apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We propose an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3 conjugated to Alexa Fluor.. 488 to discriminate G0 et M phases, respectively. The proposed method is particularly valuable in a clinical setting as verified by analyzing human leukemic cells from marrow samples or exposed to cell cycle modifiers. The method was established using the human KG1a cell line and was applied to charcterize the cell cycle and apoptosis of fresh human marrow leukemic cells. The cells were permeabilized with 1 mL of ice cold ethanol (1 h, 4°C). Following two washes with PBS, 1% FBS and 0.25% Triton X-100 (PFT), the cells were stained in 200 μL of PFT for 30 min at room temperature in the dark with 1 μg 7-AAD (Sigma-Aldrich), 5 μL Alexa Fluor.. 488-conjugated anti-human Ki67 mAb (B56, Becton-Dickinson) and 3 μL Alexa Fluor.. 488-conjugated anti-phospho(ser10)-histone H3 polyclonal antibody (Cell Signaling Technology). A control tube was prepared with 1 μg 7-AAD and 5 μL of mouse IgG1 Alexa Fluor.. 488 (Becton-Dickinson). After 2 washes with PFT, the cells were stained with 10 μL of APC-Cy7-conjugated anti-CD45 (A20, Becton-Dickinson) followed by incubation for 20 min at 4°C. Cells were then washed twice with PBS, centrifuged for 5 min at 500 g and resuspended in 300 μL of PBS. This flow cytometric method allows for a precise analysis of the impact on the cell cycle of various functional modulators. As an example, it was applied to analyze the pro-quiescent effects of contact with bone marrow MSCs as well as and the apoptosis induction and mitosis inhibition of the human KG1a leukemic cell line by camptothecin. The cell cycle characteristics of untreated KG1a cells were clearly quantified as follows: 0.4% sub-G1, 0.8% G0, 67.9% G1, 14.9% S, 14.2% G2 and 1.8% M phase. The contact with marrow MSCs during 72 h induced an increase in G0 phase and a decrease in M phase (5.3% and 0.4%, respectively). We verified the anti-proliferative and pro-apoptotic effects of 24 h exposure to camptothecin, which induced a decrease in S, G2 and M phases (6.1%, 6.2% and 0.4%, respectively) and an increase in sub-G1 phase (1.7%). Moreover, it is interesting to note that the staining protocol preserved the integrity of the plasma membrane and allows for the analysis of heterogeneous cell populations. We document here the successful utilization of this method to discriminate concomitantly apoptosis and the cell cycle phases in a model of leukemic cells exposed to inducers of cell cycle perturbations. The value of this method to analyze heterogeneous populations was shown by discriminating the marrow cells from acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals An advanced cell cycle tag toolbox reveals principles underlying temporal control of structure-selective nucleases

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Julia Bittmann ◽  
Rokas Grigaitis ◽  
Lorenzo Galanti ◽  
Silas Amarell ◽  
Florian Wilfling ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cell Cycle Regulation ◽  
Cell Cycle Control ◽  
S Phase ◽  
Specific Cell ◽  
Protein Functionality ◽  
M Phase ◽  
Cell Cycle Phases ◽  
Cycle Regulation

Cell cycle tags allow to restrict target protein expression to specific cell cycle phases. Here, we present an advanced toolbox of cell cycle tag constructs in budding yeast with defined and compatible peak expression that allow comparison of protein functionality at different cell cycle phases. We apply this technology to the question of how and when Mus81-Mms4 and Yen1 nucleases act on DNA replication or recombination structures. Restriction of Mus81-Mms4 to M phase but not S phase allows a wildtype response to various forms of replication perturbation and DNA damage in S phase, suggesting it acts as a post-replicative resolvase. Moreover, we use cell cycle tags to reinstall cell cycle control to a deregulated version of Yen1, showing that its premature activation interferes with the response to perturbed replication. Curbing resolvase activity and establishing a hierarchy of resolution mechanisms are therefore the principal reasons underlying resolvase cell cycle regulation.

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