Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron microscopy

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1263-1270
Author(s):  
M. Biggiogera ◽  
K. Burki ◽  
S.H. Kaufmann ◽  
J.H. Shaper ◽  
N. Gas ◽  
...  
Keyword(s):  
Silver Staining ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Dense Fibrillar Component ◽  
Cell Stage ◽  
Nucleolar Activity ◽  
Morula Stage ◽  
Protein B23 ◽  
Ultrathin Sections ◽  
Fibrillar Component

The ultrastructural distribution of proteins B23 and nucleolin in the nucleolus of mouse embryos from the zygote to the early blastocyst has been analyzed by means of specific antibodies and immunocytochemistry using colloidal gold complexes as markers. In parallel, silver staining of nucleoli was carried out on ultrathin sections. Our results show that the compact prenucleolar bodies at 1- and 2-cell stage as well as the compact residual fibrillar masses observed up to the morula stage, are labelled with the two antibodies. These masses, however, are not stained with silver up to the 4-cell stage. In well-developed nucleoli, the two antibodies co-localize in the dense fibrillar component (DFC) and the granular component (GC) while fibrillar centers (FCs) are devoid of label. On the contrary, silver staining occurs in the FCs and DFC but not in the GC. Our observations suggest that there is no direct relationship between the occurrence of silver staining and the distribution of protein B23 or nucleolin. Moreover, neither the localization of the two above proteins nor silver staining are unequivocally related to the nucleolar activity.

scholarly journals Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus.

1989 ◽  
Vol 37 (9) ◽  
pp. 1371-1374 ◽  
Author(s):  
M Biggiogera ◽  
S Fakan ◽  
S H Kaufmann ◽  
A Black ◽  
J H Shaper ◽  
...  
Keyword(s):  
Hela Cell ◽  
Immunoelectron Microscopy ◽  
Silver Staining ◽  
Dense Fibrillar Component ◽  
Specific Antibodies ◽  
Granular Component ◽  
Protein B23 ◽  
Reported Data ◽  
Fibrillar Component

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.

182 EXPRESSION PATTERN OF EMBRYONIC STEM CELL-SPECIFIC microRNAs IN MOUSE PREIMPLANTATION EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 190
Author(s):  
T.-Y. Fu ◽  
P.-C. Tang
Keyword(s):  
Expression Pattern ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Cell Stage ◽  
Morula Stage

The endogenous non-coding microRNAs (miRNAs) of 18–25 nucleotides (nt) have been shown to involve in a wide variety of cellular processes as the posttranscriptional regulators by repression of translation or cleavage of mRNAs. In mammals, there are approximately 250 miRNAs that have been identified, and the cluster of miRNA-290 s (miR-290 s) has been demonstrated to express dramatically from the 2-cell to the 4-cell stage in mouse embryos examined from oocytes to the 8-cell stage. The association of miR-290 to 295 with pluripotency has been reported according to their specific expression in embryonic stem (ES) cells. It is interesting to explore the roles of these ES cell-specific miRNAs during the preimplantation stages and early differentiation at the blastocyst stage. Therefore, the objective of this study was to profile the expression pattern of ES cell-specific miRNAs (miR-291-5p, miR-293-3p, and miR-294-3p) from the 4-cell, 8- to 16-cell, morula, and blastocyst stages of mouse embryos. CD-1 F1 embryos at various developmental stages were collected from superovulated and naturally mated CD-1 mice. Total miRNAs of each stage analyzed were collected from 3 embryos for every replicate. Real-time RT-PCR was performed by using the specific stem-loop primers and the embryo lysate as template, which was prepared by heating in 4 μL of PBS at 95°C. Additionally, the in situ expressions of miR-291-5p, miR-293-3p, and miR-294-3p in mouse preimplantation embryos were confirmed by LNA™ probes specific for individual miRNAs. The embryo was fixed with 4% paraformaldehyde for 2 h at room temperature, followed by 3 times wash in PBST (0.1% TritonX-100 in PBS). After hybridization with individual 5′-fluorescein-labeled LNA™ probe, the embryo was washed with 0.1 × SSC, 2 × SSC, and TN buffer (0.1 m Tris-HCl, pH 7.5, 0.15 m NaCl) subsequently. The in situ expressions of miRNAs were detected by immunocytochemical reaction. The results indicated that the expressions of miR-291-5p, miR-293-3p, and miR-294-3p were up-regulated from the 4-cell to the morula stage and then down-regulated afterwards. It was found that the signals of miR-293-5p in an expanded blastocyst were weaker than those at the early blastocyst stage. However, it showed that the intensity of expression at the morula stage was 2 to 4 folds higher compared to that at the 4-cell stage in each miRNA analyzed. Also, the result showed that the ES cell-specific miRNAs examined were expressed in all cells in a blastocyst, i.e. tropectoderm and inner cell mass. In conclusion, we have established the expression profile of ES cell-specific miRNAs during preimplantation stages in mouse embryos. The specific roles of these miRNAs would be further investigated in the short future.

scholarly journals Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts

Reproduction ◽  
2013 ◽  
Vol 146 (2) ◽  
pp. 145-153 ◽  
Author(s):  
Sojung Kwon ◽  
Areum Kwak ◽  
Hyejin Shin ◽  
Soyoung Choi ◽  
Soohyun Kim ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Hela Cells ◽  
Protein Delivery ◽  
Cancer Cell Line ◽  
Preimplantation Embryos ◽  
Cervical Cancer Cell Line ◽  
Cell Stage ◽  
Cell Permeable Peptide ◽  
Morula Stage ◽  
Punctate Pattern

Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells, a cervical cancer cell line. The entry of LDP12–EGFP was partially blocked by amiloride, while cytochalasin D or methyl-β-cyclodextrin slightly increased the uptake. LDP12–EGFP shows efficient transduction in mouse blastocysts, but not in oocytes, two-cell-stage, or morula-stage-preimplantation embryos. LDP12-mediated delivery of EGFP–LC3, a widely used marker of autophagic activation, is successful in HeLa cells and mouse blastocysts, as it enters cells and exhibits a signature punctate pattern. The lipidation of EGFP–LC3 also normally occurs after transduction, suggesting that the transduced protein retains the functional characteristics. Collectively, we show that LDP12-driven protein delivery is a fast and convenient method applicable to mouse blastocysts and reproductive cancer cells.

Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 147-158 ◽  
Author(s):  
Shangdan Xu ◽  
Jibak Lee ◽  
Masashi Miyake
Keyword(s):  
Western Blotting ◽  
Contact Region ◽  
Relative Concentration ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Homogeneous Distribution ◽  
Mrna Levels ◽  
Cell Stage ◽  
Specific Expression ◽  
Morula Stage

SummaryExpression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α− was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α− throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

scholarly journals Delayed and stage specific phosphorylation of H2AX during preimplantation development of γ-irradiated mouse embryos

Reproduction ◽  
2007 ◽  
Vol 133 (2) ◽  
pp. 415-422 ◽  
Author(s):  
Satish Kumar Adiga ◽  
Megumi Toyoshima ◽  
Tsutomu Shimura ◽  
Jun Takeda ◽  
Norio Uematsu ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Early Stage ◽  
In Utero ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Γ Irradiation ◽  
Cell Stage ◽  
Focus Formation ◽  
Damage Site ◽  
Stage Of Development

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated in the serine 139 residue at the damage site. The phosphorylated H2AX, designated as γ-H2AX, is visible as nuclear foci in the irradiated cells which are thought to serve as a platform for the assembly of proteins involved in checkpoint response and DNA repair. It is known that early stage mammalian embryos are highly sensitive to radiation but the mechanism of radiosensitivity is not well understood. Thus, we investigated the damage response of the preimplantation stage development by analyzing focus formation of γ-H2AX in mouse embryos γ-irradiated in utero. Our analysis revealed that although H2AX is present in early preimplantation embryos, its phosphorylation after 3 Gy γ-irradiation is hindered up to the two cell stage of development. When left in utero for another 24–64 h, however, these irradiated embryos showed delayed phosphorylation of H2AX. In contrast, phosphorylation of H2AX was readily induced by radiation in post-compaction stage embryos. It is possible that phosphorylation of H2AX is inefficient in early stage embryos. It is also possible that the phosphorylated H2AX exists in the dispersed chromatin structure of early stage embryonic pronuclei, so that it cannot readily be detected by conventional immunostaining method. In either case, this phenomenon is likely to correlate with the lack of cell cycle arrest, apoptosis and high radiosensitivity of these developmental stages.

The effect of the nucleocytoplasmic ratio on protein synthesis and expression of a stage-specific antigen in early cleaving mouse embryos

Development ◽  
1987 ◽  
Vol 99 (4) ◽  
pp. 481-491 ◽  
Author(s):  
U. Petzoldt ◽  
A. Muggleton-Harris
Keyword(s):  
Protein Synthesis ◽  
Specific Antigen ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
Specific Gene ◽  
Cell Stage ◽  
Two Dimensional Gel Electrophoresis ◽  
Morula Stage ◽  
Early Mouse ◽  
Mouse Eggs

The nucleocytoplasmic ratio of fertilized mouse eggs was manipulated by removing or injecting cytoplasm by micropipette, and bisection of denuded eggs to obtain both pronuclei in one half of the eggs cytoplasm. The experimental eggs were capable of cleavage to the morula stage and, in some instances, developed to the blastocyst stage similar to unmanipulated eggs. The removal of large quantities of cytoplasm by micropipette and injecting them into a recipient egg did not provide sufficient numbers of viable eggs, whereas transfer of smaller quantities (about a quarter of the cytoplasm) was less deleterious, at least for recipient eggs. However, the alteration of the nucleocytoplasmic ratio by this method was not of the correct magnitude for the purpose of this experiment. Therefore, bisection was the preferred method whereby the nucleocytoplasmic ratio was doubled. This resulted in both pronuclei residing in one half of the egg's cytoplasm. Half eggs with one pronucleus (haploid) but retaining a nucleocytoplasmic ratio similar to unmanipulated control eggs served as additional controls for the bisection experiments. Protein synthesis was analysed by two-dimensional gel electrophoresis, showing that the 2-cell- and 4-cell-stage bisected embryos with double and normal nucleocytoplasmic ratio expressed equivalent protein synthesis patterns as control embryos of the same stage. Likewise, the stage-specific surface antigen SSEA-1 did not appear before the 6- to 8-cell stage. Also in cytoplasm transfer experiments, there was no indication that altering the nucleocytoplasmic ratio in either direction changed the timing of stage-specific gene expression. These results support the idea that stage-specific gene activity during early mouse cleavage might proceed in parallel to DNA replication cycles and is independent of the nucleocytoplasmic ratio.

Cell membrane regionalization in early mouse embryos as demonstrated by 5'-nucleotidase activity

Development ◽  
1982 ◽  
Vol 69 (1) ◽  
pp. 115-126
Author(s):  
L. Izquierdo ◽  
C. Ebensperger
Keyword(s):  
Enzyme Activity ◽  
Cell Membrane ◽  
External Surface ◽  
Enzyme Reaction ◽  
Mouse Embryos ◽  
Nucleotidase Activity ◽  
Cell Stage ◽  
Morula Stage ◽  
Selective Retention ◽  
Early Mouse

The distribution of 5'-nucleotidase activity in pre-implantation mouse embryos is studied by means ofa cytochemical method adapted from Uusitalo & Karnovsky (1977). The enzyme activity is detected, from the4-cell stage up to the morula stage, on discrete patches of the cell membane between blastomeres. Appropriatecontrols show that this distribution is not a localization artifact due to selective retention of the enzyme reaction product in the narrow interblastomeric spaces. In early blastocysts, as the blastocoel expands the enzyme activity on its lining disappears. The external surface of the trophectoderm in early blastocysts lacks any enzyme activity, whereas in late blastocysts a strong enzyme activity is detected at the embryonic trophectoderm, decreasing in intensity towards the opposite pole of the embryo. These results are compared to previous observations by other authors and the differences are mainly ascribed to differences in the cytochemical procedure employed. We conclude that during cleavage a gradual cell membrane regionalization unfolds, revealing a pattern that may be related to morphogenesis; in particular, to the localization of zonular tight junctions around the peripheral blastomeres of the morula (Izquierdo, 1977; Izquierdo, López & Marticorena, 1980).

Ago2 and GW182 expression in mouse preimplantation embryos: a link between microRNA biogenesis and GW182 protein synthesis

2010 ◽  
Vol 22 (4) ◽  
pp. 634 ◽  
Author(s):  
Xing-Hui Shen ◽  
Young-Joon Han ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim
Keyword(s):  
Protein Synthesis ◽  
Internal Standard ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Internal Reference ◽  
Cell Stage ◽  
Transcript Levels ◽  
Microrna Biogenesis

MicroRNA-mediated RNA interference appears to play a role in early development and differentiation processes in preimplantation embryos. However, the expression of its key effectors, including Ago2, a key component of the RNA-induced silencing complex, and GW182, a critical component of GW bodies (GWBs), has not been assessed in preimplantation embryos. To characterise the roles of Ago2 and GW182 in early embryo development, we determined their transcription and protein synthesis in mouse embryos. Transcript levels of Ago2 and GW182 increased steadily from the one-cell stage through to the blastocyst stage when data were not normalised against an internal reference. However, when normalised against the internal standard, transcript levels for both genes were highest in four-cell stage embryos and decreased steadily through to the blastocyst stage. Indirect immunocytochemistry showed that both AGO2 and GW182 proteins were expressed in each stage in the early embryo and were observed to colocalise in the morula and blastocyst stages. Specific silencing of mRNA expression by short interference (si) RNA against Ago2 or Dicer1 decreased the expression of selected apoptosis- and development-related microRNAs, but did not inhibit development up to the blastocyst stage. However, transcription levels of Oct3/4, Nanog and Sox2 were decreased in both Ago2- and Dicer1-knockdown embryos at the blastocyst stage. Furthermore, although knockdown of these genes did not change transcript levels of GW182, GW182 protein synthesis was decreased in blastocyst stage embryos. These results suggest that Ago2 and Dicer1 regulate GW182 protein expression in mouse embryos, which is linked to microRNA biogenesis and likely to be important for differentiation in the blastocyst stage.

scholarly journals Characterization of zygotic genome activation-dependent maternal mRNA clearance in mouse

2019 ◽  
Vol 48 (2) ◽  
pp. 879-894 ◽  
Author(s):  
Qian-Qian Sha ◽  
Ye-Zhang Zhu ◽  
Sen Li ◽  
Yu Jiang ◽  
Lu Chen ◽  
...  
Keyword(s):  
De Novo ◽  
Mouse Embryos ◽  
Developmental Competence ◽  
Preimplantation Embryos ◽  
Zygotic Genome Activation ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Maternal Transcripts ◽  
Genome Activation ◽  
Zygotic Genome

Abstract An important event of the maternal-to-zygotic transition (MZT) in animal embryos is the elimination of a subset of the maternal transcripts that accumulated during oogenesis. In both invertebrates and vertebrates, a maternally encoded mRNA decay pathway (M-decay) acts before zygotic genome activation (ZGA) while a second pathway, which requires zygotic transcription, subsequently clears additional mRNAs (Z-decay). To date the mechanisms that activate the Z-decay pathway in mammalian early embryos have not been investigated. Here, we identify murine maternal transcripts that are degraded after ZGA and show that inhibition of de novo transcription stabilizes these mRNAs in mouse embryos. We show that YAP1-TEAD4 transcription factor-mediated transcription is essential for Z-decay in mouse embryos and that TEAD4-triggered zygotic expression of terminal uridylyltransferases TUT4 and TUT7 and mRNA 3′-oligouridylation direct Z-decay. Components of the M-decay pathway, including BTG4 and the CCR4-NOT deadenylase, continue to function in Z-decay but require reinforcement from the zygotic factors for timely removal of maternal mRNAs. A long 3′-UTR and active translation confer resistance of Z-decay transcripts to M-decay during oocyte meiotic maturation. The Z-decay pathway is required for mouse embryo development beyond the four-cell stage and contributes to the developmental competence of preimplantation embryos.

scholarly journals Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin

Reproduction ◽  
2003 ◽  
pp. 111-118 ◽  
Author(s):  
BD Higgins ◽  
MT Kane
Keyword(s):  
Gene Activation ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Sodium Dependent ◽  
Morula Stage ◽  
Zygotic Gene ◽  
Zygotic Gene Activation ◽  
The One ◽  
Inositol Uptake

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.

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