scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

scholarly journals AGS3-dependent trans-Golgi network membrane trafficking is essential for compaction in mouse embryos

2020 ◽  
Vol 133 (23) ◽  
pp. jcs243238
Author(s):  
Zheng-Wen Nie ◽  
Ying-Jie Niu ◽  
Wenjun Zhou ◽  
Dong-Jie Zhou ◽  
Ju-Yeon Kim ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Contact ◽  
Protein Signaling ◽  
Cell Stage ◽  
Trans Golgi Network ◽  
Early Mouse ◽  
Golgi Network

ABSTRACTActivator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.

scholarly journals 255EXPRESSION OF TRANSCRIPTION FACTORS PRIOR TO THE MATERNAL-TO-ZYGOTIC TRANSITION IN BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 247
Author(s):  
C. Vigneault ◽  
S. McGraw ◽  
G. Bujold ◽  
M.-A. Sirard
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
Transcript Levels ◽  
Significant Difference ◽  
Maternal To Zygotic Transition ◽  
La Jolla ◽  
Triton X

During the first stages of bovine embryonic development, until the 8- to 16-cell stage, the zygote is maintained by the mRNA and proteins stored in the oocyte. New embryonic transcription is reported to begin only at the 8- to 16-cell stage even if some minor transcription is detected from the 2-cell stage. In order for this to occur, several factors are required to remodel the chromatin and activate the transcription machinery. Some regulating transcription factors are possibly present in the oocyte in their mRNA form, and their translation could enhance the maternal-to-zygotic transition (MZT). In our study, we observed the expression patterns of five transcription factors (ATF2, HMGN2, HMGB2, HUEL and MSY2) in bovine in vitro-produced embryos. Embryos were produced in vitro using selected cumulus-oocyte complexes from 3-5-mm follicles of slaughterhouse ovaries. Pooled GV or MII oocytes, and 2-, 4-, 8-cell and blastocyst-stage embryos (n=40/stage) were washed in PBS and frozen at −80°C. Each pool was spiked with 1 pg of GFP RNA containing a poly(A) tail. The RNA was extracted using the Absolutely RNA Microprep Kit (Stratagene, La Jolla, CA, USA), co-precipitated with linear acrylamide (Ambion, Austin, TX, USA) and reverse-transcribed with Omniscript (Quiagen). The quantitative amplification of the transcription factors was performed in triplicate using the equivalent of 1 oocyte or embryo per reaction on a Lightcycler (Roche, Indianapolis, IN, USA). Data were normalized with the GFP levels found in each pool and a Least-Significant-Difference method was used for statistical analysis. Immunocytochemistry studies were performed on oocytes and embryos fixed and permeabilized in a solution of paraformaldehyde and Triton X-100, and results were observed on a confocal microscope. Our results show that the transcripts of the transcription factors studied are found at higher levels in pre-MZT embryos and at lower levels in subsequent stages. For HMGN2 and MSY2, there is a decrease in mRNA during oocyte maturation. For both genes, the residual mRNA remains constant up to the 4-cell stage before another loss in transcript levels in the 8-cell stage. In the case of ATF2, HMGB2 and HUEL, the maternal transcript levels are maintained until the 4-cell stage, suggesting that the mRNA is protected from degradation until its possible translation at the MZT. These results, combined to immunolocalization of the proteins, suggest a possible implication of some of these factors in the bovine MZT.

scholarly journals Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

2017 ◽  
Author(s):  
Shai R. Joseph ◽  
Máté Pálfy ◽  
Lennart Hilbert ◽  
Mukesh Kumar ◽  
Jens Karschau ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Transcription Factor Binding ◽  
Factor Binding ◽  
Zygotic Transcription ◽  
Maternal To Zygotic Transition ◽  
Nucleosome Density ◽  
Core Histones ◽  
Genome Activation

SUMMARYUpon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

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.

Compensatory responses of protein import and transcription factor expression in mitochondrial DNA defects

2004 ◽  
Vol 286 (4) ◽  
pp. C867-C875 ◽  
Author(s):  
Anna-Maria Joseph ◽  
Arne A. Rungi ◽  
Brian H. Robinson ◽  
David A. Hood
Keyword(s):  
Transcription Factor ◽  
Mitochondrial Dna ◽  
Transcription Factors ◽  
Mitochondrial Biogenesis ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Yellow Fluorescent Protein ◽  
Nuclear Gene ◽  
Protein Levels

Defects in mitochondrial DNA (mtDNA) evoke distinctive responses in the nuclear genome, leading to altered mitochondrial biogenesis. We used C2C12 cells depleted of mtDNA (rho– cells) and fibroblasts from a mitochondrial encephalopathy, lactic acidosis, and strokelike episodes (MELAS) patient to examine adaptations of the protein import machinery and transcription factors involved in mitochondrial biogenesis. In rho– cells, Tom20 and Tim23 protein levels were reduced by 25% and 59%, whereas mtHSP70 was induced by twofold relative to control cells. These changes were accompanied by a 21% increase in enhanced yellow fluorescent protein (EYFP) import into mitochondria in rho– cells ( P < 0.05). In contrast, in MELAS cells mtHSP70 was elevated by 70%, whereas Tom20 and Tom34 protein levels were increased by 45% and 112% relative to control values. EYFP import was not altered in MELAS cells. In rho– cells, protein levels of the transcription factors nuclear respiratory factor-1 (NRF-1) and transcription factor A (Tfam) declined by 33% and 54%, whereas no change was observed for the coactivator peroxisome proliferator receptor-γ coactivator-1α (PGC-1α). In contrast, Tfam was increased by 40% in MELAS cells. Rho– cells displayed reduced oxygen consumption (V̇o2) and ATP levels, along with a twofold increase in lactate levels ( P < 0.05). In electrically stimulated C2C12 cells, 109%, 78%, 60%, and 67% increases were observed in mtDNA, V̇o2, cytochrome- c oxidase (COX) activity, and Tom34 levels, respectively ( P < 0.05). Our findings suggest that compensatory adaptations occurred to maintain normal rates of protein import in response to mtDNA defects and support a role for contractile activity in reducing pathophysiology associated with mtDNA depletion. Because the expression of nuclear-encoded transcription factors and protein import machinery components was dependent on the type of mtDNA defect, these findings suggest involvement of distinct signaling cascades, each dependent on the type of mitochondrial defect, resulting in divergent changes in nuclear gene expression patterns.

scholarly journals EBR1, a Novel Zn2Cys6 Transcription Factor, Affects Virulence and Apical Dominance of the Hyphal Tip in Fusarium graminearum

2011 ◽  
Vol 24 (12) ◽  
pp. 1407-1418 ◽  
Author(s):  
Chunzhao Zhao ◽  
Cees Waalwijk ◽  
Pierre J. G. M. de Wit ◽  
Theo van der Lee ◽  
Dingzhong Tang
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fusarium Graminearum ◽  
Apical Dominance ◽  
Radial Growth ◽  
Fluorescent Protein ◽  
Orthologous Gene ◽  
Protein Fusion ◽  
Knock Out ◽  
Hyphal Tip

Zn2Cys6 transcription factors are unique to fungi and have been reported to be involved in different regulatory functions. Here, we characterized EBR1 (enhanced branching 1), a novel Zn2Cys6 transcription factor of Fusarium graminearum. Knocking out EBR1 in F. graminearum PH-1 caused reduction of both radial growth and virulence. The conidia of knock-out strain PH-1Δebr1 germinated faster than those of wild-type PH-1, but the conidiation of the mutant was significantly reduced. Detailed analysis showed that the reduced radial growth might be due to reduced apical dominance of the hyphal tip, leading to increased hyphal branching. Inoculation assays on wheat heads with a green fluorescent protein (GFP)-labeled PH-1Δebr1 mutant showed that it was unable to penetrate the rachis of the spikelets. Protein fusion with GFP showed that EBR1 is localized in the nucleus of both conidia and hyphae. Knocking out the orthologous gene FOXG_05408 in F. oxysporum f. sp. lycopersici caused a much weaker phenotype than the PH-1Δebr1 mutant, which may be due to the presence of multiple orthologous genes in this fungus. Transformation of FOXG_05408 into PH-1Δebr1 restored the mutant phenotype. Similar to EBR1, FOXG_05408 is localized in the nucleus of F. oxysporum f. sp. lycopersici. Possible functions of EBR1 and its relation with other fungal transcription factors are discussed.

Nuclear cytoplasmic interactions following nuclear transplantation in mouse embryos

Development ◽  
1987 ◽  
Vol 101 (4) ◽  
pp. 915-923 ◽  
Author(s):  
S.K. Howlett ◽  
S.C. Barton ◽  
M.A. Surani
Keyword(s):  
Cell Nucleus ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Nuclear Transplantation ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Cell Donor ◽  
Donor Nucleus ◽  
Cell Embryo

We have investigated the development of reconstituted embryos in which enucleated 1- or 2-cell embryos received various advanced nuclei. Enucleated 1-cells developed to the blastocyst stage only when an early 2-cell donor nucleus was transferred but very rarely if the donor nucleus was derived from a late 2-cell, early 4-cell or mid 8-cell embryo. Although an 8-cell nucleus could only support development of an enucleated zygote to the 2-cell stage, it did express the hsp 68/70 X 10(3) Mr proteins that are characteristic of the first embryonic gene activity. These polypeptides were absent in enucleated zygotes that did not receive a donor nucleus. Moreover, an 8-cell nucleus transferred to an enucleated late 2-cell blastomere could also support preimplantation development provided that the nuclear:cytoplasmic ratio was maintained as in intact 2-cell blastomeres. 8-cell nuclei transferred to zygotes that retained at least one pronucleus were able to support development to the blastocyst stage provided that the pronucleus was both fully transcriptionally active and present beyond the late 1-cell stage. This study suggests an active and continued helper role of the resident pronucleus for the participation by an 8-cell nucleus in reconstituted eggs.

scholarly journals Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres

Reproduction ◽  
2005 ◽  
Vol 129 (5) ◽  
pp. 547-556 ◽  
Author(s):  
Sigrid Eckardt ◽  
N Adrian Leu ◽  
Satoshi Kurosaka ◽  
K John McLaughlin
Keyword(s):  
Somatic Cell ◽  
Fluorescent Protein ◽  
Cumulus Cell ◽  
Cell Nuclei ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Embryonic Genome ◽  
Somatic Genome ◽  
Cleavage Stages ◽  
Green Fluorescent

Mammalian somatic cell cloning requires factors specific to the oocyte for reprogramming to succeed. This does not exclude that reprogramming continues during the zygote and cleavage stages. The capacity or role of zygotic and cleavage stages to reprogram somatic cell nuclei is difficult to assess due to the limited development of somatic cell nuclei transplanted into cytoplasts of these stages. Alternatively, tetraploid embryos have been used to study reprogramming and can be assessed for their contribution to extra-embryonic lineages. When mouse cumulus cell nuclei transgenic for Oct4-green fluorescent protein (GFP) were injected into intact two- and four-cell stage blastomeres, manipulated embryos developed into blastocysts with expression of Oct4-GFP as observed in embryos produced by nuclear transfer into metaphase II oocytes. However, only the latter contributed to extra-embryonic tissues in day 10.5 conceptuses, with the exclusion of the somatic genome in cells originating from transfer into blastomeres already at 5.5 days post conception. Somatic nuclei transferred into cleavage stage blastomeres reinitiated expression of an embyronic-specific transgene, but lacked the extent of reprogramming required for contribution to postimplantation development, even when complemented by an embryonic genome.

scholarly journals Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Shai R Joseph ◽  
Máté Pálfy ◽  
Lennart Hilbert ◽  
Mukesh Kumar ◽  
Jens Karschau ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Transcription Factor Binding ◽  
Factor Binding ◽  
Zygotic Transcription ◽  
Maternal To Zygotic Transition ◽  
Nucleosome Density ◽  
Core Histones ◽  
Genome Activation

Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

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