Uridine and guanosine incorporation by the mouse one-cell embryo

Development ◽  
1978 ◽  
Vol 44 (1) ◽  
pp. 133-148
Author(s):  
R. J. Young ◽  
K. Sweeney ◽  
J. M. Bedford
Keyword(s):  
Cell Stage ◽  
Trichloracetic Acid ◽  
Insoluble Material ◽  
Embryonic Genome ◽  
Internal Position ◽  
Cell Embryo ◽  
The One ◽  
Sugar Derivatives ◽  
Hydrolysis Of ◽  
Derivatives Of

The activity of the embryonic genome prior to the first cleavage has been assessed by studying the uptake of [3H]uridine, its phosphorylation and incorporation into RNA by mouse one-cell embryos. One-cell embryos incorporated [3H]uridine linearly into cold trichloracetic acid (TCA) insoluble material at a low level 1–9 h post fertilization. The incorporation of [3H]guanosine was also low but followed a biphasic curve which had a steeper slope at 1–3 h than during the period 4–9 h post fertilization. Unfertilized mouse ova incorporated very little [3H]uridine or [3H]guanosine into TCA insoluble material, and much of this was RNase insensitive. Dimethyl sulfoxide (DMSO) enhanced the uptake of [3H]thymidine and its incorporation into pronuclear DNA by one-cell embryos, but had no effect on the incorporation of [3H]uridine by them, or of [3H] uridine and [3H]guanosine by unfertilized ova. The uptake and incorporation of [3H] guanosine by one-cell embryos were enhanced by DSMO, but only during the period 1–3 h post fertilization. Sugar derivatives of UDP, and UMP, UDP, UTP, CMP, CDP and CTP have been identified in the soluble fraction obtained from mouse one-cell embryos incubated with [3H] uridine 1–3 h post fertilization. Very little of the [3H] uridine taken up by the embryos is present as [3H] UTP, or [3H] CTP; most is found as [3H] UMP or [3H] UDP or as the sugar derivatives. Alkaline or ribonuclease (A, T1 and T2) hydrolysis of the 3H-labeled ethanol insoluble material precipitated from the lysate of one-cell embryos incubated with [3H] uridine 1–3 h post fertilization liberated radioactive cytidine and uridine-3'-phosphates. This demonstrates that [3H] uridine is incorporated into an internal position in RNA and suggests that RNA synthesis does occur in the one-cell embryo 1–3 h post fertilization. Since pronuclei of one-cell embryos incubated with [3H] uridine were not labeled it appears, however, that the RNA synthesized at the one-cell stage is not a product of the embryonic genome.

scholarly journals Human embryonic genome activation initiates at the one-cell stage

2021 ◽  
Author(s):  
Maki Asami ◽  
Brian Y.H. Lam ◽  
Marcella K. Ma ◽  
Kara Rainbow ◽  
Stefanie Braun ◽  
...  
Keyword(s):  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
The One ◽  
Genome Activation

scholarly journals cAMP-responsive element-binding protein expression and regulation in the mouse preimplantation embryo

Reproduction ◽  
2007 ◽  
Vol 134 (5) ◽  
pp. 667-675 ◽  
Author(s):  
X L Jin ◽  
C O’Neill
Keyword(s):  
Transcription Factors ◽  
Preimplantation Embryo ◽  
Binding Protein ◽  
Dependent Manner ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Element Binding Protein ◽  
Cell Embryo ◽  
Responsive Element ◽  
Camp Responsive Element Binding

Gene expression from the new embryonic genome is required for normal preimplantation embryo development. Two members of the cAMP-responsive element-binding protein (Creb) family of transcription factors, Creb1 and activating transcription factor 1 (Atf1), are essential for normal preimplantation development. These transcription factors are activated by phosphorylation. Creb1 mRNA was expressed throughout the preimplantation phase. Cytoplasmic immunolocalization of Creb1 was detected in all preimplantation embryo stages. The antigen was largely excluded from the pronuclei/nuclei at embryonic stages except in the mid-cycle two-cell and compacted eight-cell embryo. Activation-state-specific antibodies showed serine 133 phosphorylated Creb1 localization was similar to Creb1 staining, except that there was no increase in staining at the eight-cell stage. Increased staining of phosphorylated Creb1 was observed in the nucleus of mid-cycle two-cell embryos. Increased expression of phosphorylated Creb1 in the two-cell embryo was induced by brief exposure of embryos to ionomycin, but not by a dibutyryl cAMP. This was blocked by buffering intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester), but not by a cAMP antagonist, Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine. Calmodulin is an intracellular receptor for calcium. Calmodulin mRNA was expressed throughout the preimplantation phase of development. The calmodulin antagonist, W-7, inhibited the ionomycin-induced localization of phosphorylated Creb1 in the nucleus. Treatment of embryos with W-7 caused a dose-dependent inhibition of normal development of zygotes to the blastocysts stage. The study shows Creb1 expression and nuclear localization was dynamically regulated in the early embryo. The marked nuclear accumulation and phosphorylation of Creb1 at the two-cell stage occurred at the time of transcription from the embryonic genome and was regulated in a calcium- and calmodulin-dependent manner.

scholarly journals The regulation of the expression and activation of the essential ATF1 transcription factor in the mouse preimplantation embryo

Reproduction ◽  
2014 ◽  
Vol 148 (2) ◽  
pp. 147-157 ◽  
Author(s):  
X L Jin ◽  
C O'Neill
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Map Kinase ◽  
P38 Map Kinase ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Calcium Calmodulin Dependent ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Active Transcription

The co-expression of the CREB and ATF1 transcription factors is required for the development of preimplantation embryos. Embryotropin-mediated, calcium/calmodulin-dependent signalling activates CREB-induced transcription in the two-cell embryo, but the regulation of ATF1 in the embryo is not known. This study demonstrates that ATF1 begins to accumulate within both pronuclei of the mouse zygote by 20 h post-human chorionic gonadotrophin. This did not require new transcription (not blocked by α-amanitin), but was dependent upon protein synthesis (blocked by puromycin) and the activity of P38 MAP kinase. ATF1 becomes an active transcription factor upon being phosphorylated. A marked accumulation of phosphorylated ATF1 was evident in two-cell embryos and this persisted in subsequent stages of development. This phosphorylation was enhanced by the actions of autocrine embryotropic mediators (including Paf) and required the mutual actions of P38 MAP kinase and calmodulin-dependent pathways for maximum levels of phosphorylation. The combined inhibition of these two pathways blocked embryonic genome activation (EGA) and caused embryos to enter a developmental block at the two-cell stage. The members of the CREB family of transcription factors can generate one of the most diverse transcriptomes of any transcription factor. The demonstration of the presence of activated CREB and ATF1 within the embryonic nucleus at the time of EGA places these transcription factors as priority targets as key regulators of EGA.

scholarly journals FRACTIONATION OF GELATIN

1929 ◽  
Vol 12 (3) ◽  
pp. 379-390 ◽  
Author(s):  
M. Kunitz ◽  
John H. Northrop
Keyword(s):  
Dilute Hydrochloric Acid ◽  
Soluble Fraction ◽  
Insoluble Fraction ◽  
Partial Hydrolysis ◽  
Free Solution ◽  
Buffer Solution ◽  
Solution Ph ◽  
Insoluble Material ◽  
The One ◽  
Hydrolysis Of

1. It is possible to fractionate gelatin by means of reprecipitation at 23°C. of a salt-free solution of pH 4.7 into two fractions, one of which is soluble in water at any temperature, and a second one which does not dissolve in water even when heated to 80°C. 2. The proportion of the soluble fraction in gelatin is much greater than of the insoluble one. 3. The insoluble fraction of gelatin does not swell when mixed with water, but it does swell in the presence of acid and alkali which finally dissolve it. 4. Blocks of concentrated gel made by dissolving various mixtures of the soluble and insoluble fractions of gelatin in dilute NaOH swell differently when placed in large volumes of dilute buffer solution pH 4.7 at 5°C. The gel consisting of the insoluble material shows only a trace of swelling, while those containing a mixture of soluble and insoluble swell considerably. The swelling increases rapidly as the proportion of the soluble fraction increases. 5. A 5 per cent gel made up by dissolving the insoluble fraction of gelatin in dilute NaOH loses about 70 per cent of its weight when placed in dilute buffer pH 4.7 at 5°C. A similar gel made up of ordinary gelatin loses only about 20 per cent of its weight under the same conditions. 6. It was not found possible to resynthesize isoelectric gelatin from its components. 7. An insoluble substance similar in many respects to the one obtained by reprecipitation of gelatin is produce on partial hydrolysis of gelatin in dilute hydrochloric acid at 90°C.

scholarly journals The kinases PIG-1 and PAR-1 act in redundant pathways to regulate asymmetric division in the EMS blastomere of C. elegans

2017 ◽  
Author(s):  
Malgorzata J. Liro ◽  
Diane G. Morton ◽  
Lesilee S. Rose
Keyword(s):  
Wnt Pathway ◽  
Asymmetric Division ◽  
Spindle Orientation ◽  
Cell Stage ◽  
Spindle Positioning ◽  
C Elegans ◽  
Stage Embryo ◽  
Cell Embryo ◽  
The One

AbstractThe PAR-1 kinase of C. elegans is localized to the posterior of the one-cell embryo and its mutations affect asymmetric spindle placement and partitioning of cytoplasmic components in the first cell cycle. However, unlike mutations in the posteriorly localized PAR-2 protein, par-1 mutations do not cause failure to restrict the anterior PAR polarity complex. Further, it has been difficult to examine the role of PAR-1 in subsequent divisions due to the early defects in par-1 mutant embryos. Here we show that the PIG-1 kinase acts redundantly with PAR-1 to restrict the anterior PAR-3 protein for polarity maintenance in the one-cell embryo. By using a weak allele of par-1 that exhibits enhanced lethality when combined with a pig-1 mutation we have further explored roles for these genes in subsequent divisions. We find that both PIG-1 and PAR-1 regulate spindle orientation in the EMS blastomere of the four-cell stage embryo to ensure that it undergoes an asymmetric division. In this cell, PIG-1 and PAR-1 act in parallel pathways for spindle positioning, PIG-1 in the MES-1/SRC-1 pathway and PAR-1 in the Wnt pathway.

scholarly journals Modelling human zygotic genome activation in 8C-like cells in vitro

2021 ◽  
Author(s):  
Jasmin Taubenschmid-Stowers ◽  
Maria Rostovskaya ◽  
Fatima Santos ◽  
Sebastian Ljung ◽  
Ricard Argelaguet ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Human Embryos ◽  
Marker Genes ◽  
Cell Stage ◽  
Embryonic Genome Activation ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Genome Activation

The remodelling of the epigenome and transcriptome of the fertilised oocyte to establish totipotency in the zygote and developing embryo is one of the most critical processes in mammalian embryogenesis. Zygotic or embryonic genome activation (ZGA, EGA) in the 2-cell embryo in mouse, and the 8-cell embryo in humans, constitutes the first major wave of transcription. Failure to initiate ZGA leads to developmental defects, and contributes to the high attrition rates of human pre-implantation embryos. Due to limitations in cell numbers and experimental tractability, the mechanisms that regulate human embryonic genome activation in the totipotent embryo remain poorly understood. Here we report the discovery of human 8-cell like cells (8CLCs) specifically among naive embryonic stem cells, but not primed pluripotent cells. 8CLCs express ZGA marker genes such as ZSCAN4, LEUTX and DUXA and their transcriptome closely resembles that of the 8-cell human embryo. 8-cell like cells reactivate 8-cell stage specific transposable elements such as HERVL and MLT2A1 and are characterized by upregulation of the DNA methylation regulator DPPA3. 8CLCs show reduced SOX2 protein, and can be identified based on expression of the novel ZGA-associated protein markers TPRX1 and H3.Y in vitro. Overexpression of the transcription factor DUX4 as well as spliceosome inhibition increase ZGA-like transcription and enhance TPRX1+ 8CLCs formation. Excitingly, the in vitro identified 8CLC marker proteins TPRX1 and H3.Y are also expressed in 8-cell human embryos at the time of genome activation and may thus be relevant in vivo. The discovery of 8CLCs provides a unique opportunity to model and manipulate human ZGA-like transcriptional programs in vitro, and might provide critical functional insights into one of the earliest events in human embryogenesis in vivo.

The relationship between cleavage, DNA replication, and gene expression in the mouse 2-cell embryo

Development ◽  
1984 ◽  
Vol 79 (1) ◽  
pp. 139-163
Author(s):  
V. N. Bolton ◽  
P. J. Oades ◽  
M. H. Johnson
Keyword(s):  
Dna Replication ◽  
Synthetic Activity ◽  
Cleavage Division ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Embryonic Genome ◽  
Cell Embryo ◽  
Two Phases ◽  
The Relationship

The 2-cell stage of mouse embryogenesis is characterized by two phases of α-amanitin-sensitive polypeptide synthetic activity, which appear to mark the first major expression of the embryonic genome, as assessed by examination of in vitro translates of mRNA. Using populations of embryos synchronized to the first cleavage division, we have established that DNA replication takes place over the period 1 to 5·5 h after the first cleavage division; the two bursts of putative transcription take place before and immediately after DNA replication, and the translation products are detectable in each case within 3–4 h. In addition, we have shown that suppression of cytokinesis and the second round of DNA replication does not affect synthesis of the α-amanitin-sensitive polypeptides, and that neither DNA replication nor the loss of maternal mRNA that take place during the 2-cell stage are dependent upon synthesis of the α-amanitin-sensitive polypeptides.

scholarly journals Stella modulates transcriptional and endogenous retrovirus programs during maternal-to-zygotic transition

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yun Huang ◽  
Jong Kyoung Kim ◽  
Dang Vinh Do ◽  
Caroline Lee ◽  
Christopher A Penfold ◽  
...  
Keyword(s):  
Developmental Process ◽  
Endogenous Retrovirus ◽  
Endogenous Retroviruses ◽  
Cell Stage ◽  
Developmental Potential ◽  
Chimeric Transcripts ◽  
Embryonic Genome ◽  
Maternal To Zygotic Transition ◽  
Cell Embryo

The maternal-to-zygotic transition (MZT) marks the period when the embryonic genome is activated and acquires control of development. Maternally inherited factors play a key role in this critical developmental process, which occurs at the 2-cell stage in mice. We investigated the function of the maternally inherited factor Stella (encoded by Dppa3) using single-cell/embryo approaches. We show that loss of maternal Stella results in widespread transcriptional mis-regulation and a partial failure of MZT. Strikingly, activation of endogenous retroviruses (ERVs) is significantly impaired in Stella maternal/zygotic knockout embryos, which in turn leads to a failure to upregulate chimeric transcripts. Amongst ERVs, MuERV-L activation is particularly affected by the absence of Stella, and direct in vivo knockdown of MuERV-L impacts the developmental potential of the embryo. We propose that Stella is involved in ensuring activation of ERVs, which themselves play a potentially key role during early development, either directly or through influencing embryonic gene expression.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

Theoretical Study of the Process of Passage of Glycoside Amides through the Cell Membrane of Cancer Cell

2020 ◽  
Vol 20 ◽  
Author(s):  
Vasil Tsanov ◽  
Hristo Tsanov
Keyword(s):  
Cell Membrane ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Quantum Chemical ◽  
Density Functional ◽  
Enzyme Regulation ◽  
Amide Derivatives ◽  
Pass Through ◽  
Hydrolysis Of ◽  
Derivatives Of

Background:: This article concentrates on the processes occurring in the medium around the cancer cell and the transfer of glycoside amides through their cell membrane. They are obtained by modification of natural glycoside-nitriles (cyano-glycosides). Hydrolysis of starting materials in the blood medium and associated volume around physiologically active healthy and cancer cells, based on quantum-chemical semi-empirical methods, is considered. Objective:: Based on the fact that the cancer cell feeds primarily on carbohydrates, it is likely that organisms have adapted to take food containing nitrile glycosides and / or modified forms to counteract "external" bioactive activity. Cancers, for their part, have evolved to create conditions around their cells that eliminate their active apoptotic forms. This is far more appropriate for them than changing their entire enzyme regulation to counteract it. In this way, it protects itself and the gene sets and develops according to its instructions. Methods:: Derived pedestal that closely defines the processes of hydrolysis in the blood, the transfer of a specific molecular hydrolytic form to the cancer cell membrane and with the help of time-dependent density-functional quantum- chemical methods, its passage and the processes of re-hydrolysis within the cell itself, to forms causing chemical apoptosis of the cell - independent of its non-genetic set, which seeks to counteract the process. Results:: Used in oncology it could turn a cancer from a lethal to a chronic disease (such as diabetes). The causative agent and conditions for the development of the disease are not eliminated, but the amount of cancer cells could be kept low for a long time (even a lifetime). Conclusion:: The amide derivatives of nitrile glycosides exhibit anti-cancer activity, the cancer cell probably seeks to displace hydrolysis of these derivatives in a direction that would not pass through its cell membrane and the amide- carboxyl derivatives of nitrile glycosides could deliver extremely toxic compounds within the cancer cell itself and thus block and / or permanently damage its normal physiology.

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